CN102576027B

CN102576027B - Imaging and evaluating embryos, oocytes, and stem cells

Info

Publication number: CN102576027B
Application number: CN201080047415.4A
Authority: CN
Inventors: C.C.王; K.E.洛克; T.M.贝尔; R.A.赖乔-佩拉; B.贝尔
Original assignee: Leland Stanford Junior University
Current assignee: Leland Stanford Junior University
Priority date: 2009-08-22
Filing date: 2010-08-23
Publication date: 2014-10-01
Anticipated expiration: 2030-08-23
Also published as: EA025172B9; US20170089820A1; ZA201201363B; HK1166521A1; EP2827150B3; EP3805762B1; CN102576027A; IL218239A; AU2010286740B2; US20120095287A1; PL2827150T6; EA201270300A1; SG178536A1; IN2012DN02452A; MX2012002266A; EP2430454B1; US20110092762A1; US7963906B2; EP2615460B1; PT2827150T

Abstract

Methods, compositions and kits for determining the developmental potential of one or more embryos or pluripotent cells and/or the presence of chromosomal abnormalities in one or more embryos or pluripotent cells are provided. These methods, compositions and kits find use in identifying embryos and oocytes in vitro that are most useful in treating infertility in humans.

Description

Imaging is also assessed embryo, egg mother cell and stem cell

The cross reference of related application

The application requires the U.S. Provisional Patent Application the 61/332nd of submitting on May 7th, 2010, the U.S. Provisional Patent Application the 61/236th that No. 651 and on August 22nd, 2009 submit to, and the right of priority of No. 085, both are incorporated to this paper by reference of text for it.

Invention field

The present invention relates to from the biology of both zygote/embryos of human and animal, egg mother cell and stem cell and the field of clinical trial and especially imaging and assessment.

Background of invention

Sterility is the common health problem that affects the Mr. and Mrs of 10-15% childbearing age.2006 are only in the U.S., have approximately carried out (IVF) in vitro fertilization in 140,000 cycles (cdc.gov/art).This produces and exceedes every year 1000000 for implanting and growing the mature variable and usually embryo's of unclear potentiality the cultivation that has.Tire rate alive after IVF, each cycle be only 29%, and average 30% tire alive causes multifetation (cdc.gov/art).Multifetation all has the sufficient unfavorable result of evidence, for example miscarriage, premature labor and low birthrate to mother and fetus.The possible cause of IVF failure is different; But since within 1978, introducing IVF, one of main challenge is to identify the embryo who is suitable for transfer and most probable generation full-term pregnancy most.

Cognition in embryonic development field, basis is restricted, because biological research still has dispute and usually exempts from research fund to human embryos.As a result, the major part in the existing knowledge of embryonic development derives from the research of model organism.But in the time experiencing similar developmental stage from the embryo of different plant species, opportunity is because of species difference.These differences and many other differences make it be unsuitable for directly knowing another species (Taft, R.E. (2008) Theriogenology 69 (1): 10-16) by inference from species.The general way that the mankind grow, and basic basic molecule determinative, it is unique growing for human embryos.For example, in mouse, embryo transcribes at after fertilization and approximately within 12 hours, is activated, with the First cleavage division while, and in human embryos, embryonic gene activation (EGA) occurs in the 3rd day, approximately 8 cell period (Bell, the people such as C.E. (2008) Mol.Hum.Reprod.14:691-701; Braude, the people such as P. (1988) Nature 332:459-461; Hamatani, the people such as T. (2004) Proc.Natl.Acad.Sci.101:10326-10331; Dobson, the people such as T. (2004) Human Molecular Genetics 13 (14): 1461-1470).In addition the gene that, the mankind are conditioned in growing is in early days unique (Dobson, the people such as T. (2004) Human Molecular Genetics 13 (14): 1461-1470).And, at other species for example in mouse, the embryo who exceedes 85% in vitro culture reaches blastocyst stage, one of developmental first boundary mark of mammal, and the human embryos of cultivating have the average blastocyst formation rate of about 30-50%, and high rate (Rienzi, the people such as L. (2005) the Reprod.Biomed.Online 10:669-681 of mosaicism and for example fragmentation of undesired phenotype and arrest of development; Alikani, the people such as M. (2005) Mol.Hum.Reprod.11:335-344; Keltz, the people such as M.D. (2006) Fertil.Steril.86:321-324; French, the people such as D.B. (2009) Fertil.Steril.).Although there are these differences, most PIE is grown research and is derived from model organism and be difficult to and human embryos related to development (Zernicka-Goetz, M. (2002) Development 129:815-829; Wang, the people such as Q. (2004) Dev Cell.6:133-144; Bell, the people such as C.E. (2008) Mol.Hum.Reprod.14:691-701; Zernicka-Goetz, M. (2006) Curr.Opin.Genet.Dev.16:406-412; Mtango, the people such as N.R. (2008) Int.Rev.Cell.Mol.Biol.268:223-290).

Traditionally in IVF is clinical, by simple morphological observation, for example evenly existence and the cytoclastic degree of blastomere size, monokaryon are measured (Rijinders PM, Jansen CAM. (1998) Hum Reprod 13:2869-73 to human embryos viablity; People (2002) the Fertil Steril77:1191-5 such as Milki AA).Recently, such as embryo's of other method Extending culture (to the blastocyst stage of the 5th day) and be used to measure embryo quality (people (2000) the Fertil Steril 73:126-9 such as Milki A via the chromosome analysis of preimplantation genetic diagnosis (PGD); Fragouli E, the EPub before (2009) Fertil Steril Jun 21[printing and publishing]; People (2009) the Hum Reprod 6:20 such as El-Toukhy T; People (2009) the Nat Med 15:577-83 such as Vanneste E).But the potential risk of these methods also exists, because their Extending culture phases and destroy embryo integrality (people (2009) the Fertil Steril91:305-15 such as Manipalviratn S; People (2007) the N Engl J Med.357:9-17 such as Mastenbroek S).

Recently shown is that time difference imaging can be the useful instrument of observing early embryonic development.Certain methods has been used injection (ICSI) human embryos afterwards in time difference Imaging: Monitoring sperm archiblast to grow (people (1994) Human Reproduction.9 (9): the 1743-1748 such as Nagy; The people such as Payne (1997) Human Reproduction.12:532-541).The 3rd day, analyze polar body discharge and Pronucleus formation and be associated with good morphology.But, the not parameter relevant with blastocyst formation or pregnant result.Additive method has been paid close attention to as the beginning of the First cleavage of instruction with predict human embryo's viablity (people (2002) the Human Reproduction such as Fenwick, 17:407-412; The people such as Lundin (2001) Human Reproduction 16:2652-2657).But these methods are not approved the importance in the time interval between duration or the early stage division of cytokinesis.

Fissional opportunity and degree (WO/2007/144001) during additive method has used time difference imaging with measurement early embryonic development.But these methods are the fundamental sum conventional method of the open time difference imaging for ox embryo only, near its opportunity in developmental potentiality, morphologic features, molecule and rear raw program and shifting is substantially different from human embryos with in parameter.For example, ox embryo spends the much longer time and implants (being respectively 30 days and 9 days) compared with human embryos.(Taft,(2008)Theriogenology69(1):10-16)。And, less than specific imaging parameters or the time interval that openly can estimate human embryos viablity.

Recently, time difference imaging has been used to observe human embryos during initial 24 hours after fertilization and has grown people (2008) Reproductive BioMedicine Online17 (3): 385-391 such as () Lemmen.Find that division is first afterwards nuclear synchronous relevant with pregnant result.But this work early stage spilting of an egg first of reaching a conclusion is not important expectation parameter, it contradicts (people (2002) the Human Reproduction17:407-412 such as Fenwick with research before; The people such as Lundin (2001) Human Reproduction16:2652-2657).

Finally, do not study by being associated and confirming imaging parameters with embryo's point subroutine or embryo's chromosomal.Therefore aspect some, lack the method for human embryos assessment and can improve by this method, it comprises the relevance of new application, graphical analysis and imaging parameters and molecular spectra and the chromosomal of time difference microscopy.The present invention addresses these problems.

CN101495619A relates to a kind of embryo quality appraisal procedure based on blastomere division and motion.

Summary of the invention

Method, composition and kit for determining one or more embryos or one or more embryos' multipotential cell or the developmental potentiality of multipotential cell are provided.These methods, composition and kit determine to have in the embryo of ability (ability) that good developmental potentiality bud into blastocyst or ability (capacity) and egg mother cell usefully in vitro, and therefore it be useful in method of the sterility in the treatment mankind and similar approach.

In aspect more of the present invention, provide the method for the developmental potentiality for determining embryo or multipotential cell.In these areas, measure one or more cell parameterses of embryo or multipotential cell to obtain cell parameters measurement result.Be used to provide determining of developmental potentiality to described embryo or multipotential cell after cell parameters, described determine can be used for instructing clinical course of action.In some embodiments, cell parameters is by the measurable morphology event of time difference microscopy.In some embodiments, for example, in the time that embryo is determined, described one or more cell parameterses are: cytokinesis event is the duration of cytokinesis 1 for example; The time interval between the time interval between cytokinesis 1 and cytokinesis 2 and cytokinesis 2 and cytokinesis 3.In certain embodiments, the duration of cell cycle 1 is also as cell parameters.In some embodiments, cell parameters measurement result is by comparing it and providing the definite of described embryonic development potentiality to use by this result relatively with the comparable cell parameters measurement result of carrying out self-reference embryo.In some embodiments, embryo is human embryos.In some embodiments, cell parameters is measured to obtain the gene expression dose of gene expression measurement result.In some embodiments, gene expression measurement result by by its with come self-reference multipotential cell or embryo or compare to use from its gene expression measurement result of one or more cells, wherein this result is relatively used to provide described multipotential cell or embryo's the determining of developmental potentiality.In some embodiments, described embryo is human embryos.

In aspect more of the present invention, be provided for by embryo or multipotential cell according to them the method with respect to the developmental potentiality classification of other embryos or multipotential cell in group.In these embodiments, one or more cell parameterses of measuring described embryo in described group or multipotential cell are to obtain each cell parameters measurement result of described embryo or multipotential cell.Each of embryo or multipotential cell described in afterwards described cell parameters measurement result being used for to determine described group developmental potentiality relative to each other, described definite clinical course of action that can be used for instructing.In some embodiments, cell parameters is by the measurable morphology event of time difference microscopy.In some embodiments, for example, in the time that embryo is graded, one or more cell parameterses are for example duration of cytokinesis 1 of cytokinesis event; The time interval between the time interval between cytokinesis 1 and cytokinesis 2 and cytokinesis 2 and cytokinesis 3.In certain embodiments, the duration of cell cycle 1 is also measured.In some embodiments, cell parameters is the expression of one or more genes.In some embodiments, one or more cell parameters measurement results are by being compared to each other to determine described embryo or multipotential cell developmental potentiality relative to each other by the described embryo from described group or each the described cell parameters measurement result in multipotential cell.In some embodiments, described one or more cell parameters measurement result compares to determine that by each cell parameters measurement result and the cell parameters measurement result of coming self-reference embryo or multipotential cell being compared to determine from the developmental potentiality of each embryo or multipotential cell and by these developmental potentialities described embryo or multipotential cell developmental potentiality relative to each other uses.

In aspect more of the present invention, be provided for being provided for for supplementary reproduction (IVF) embryo's with good developmental potentiality who shifts to jenny method.In these areas, under condition sufficient for embryonic development, cultivate one or more embryos.In described one or more embryos, measure afterwards one or more cell parameterses to obtain cell parameters measurement result.Afterwards by described cell parameters measurement result for provide described one or more embryos developmental potentiality determine.The one or more embryos that confirm good developmental potentiality are transferred in jenny afterwards.

Accompanying drawing summary

In the time reading in conjunction with appended accompanying drawing, understand best the present invention from following detailed description.Emphasize, according to convention, the different characteristic of accompanying drawing is not to scale (NTS).On the contrary, for the diameter of knowing different characteristic zooms in or out arbitrarily.Accompanying drawing comprises following each figure.

Fig. 1 is the process flow diagram showing for assessment of embryo.

Fig. 2 shows the cell spilting of an egg of time period and a series of photos of division of 6 days.Image is indicated the 1st day to the 6th day.Engineer's scale represents 50 μ m.

Fig. 3 shows from 1 cell stage (zygote) successfully to grow the histogram for the number percent of blastocyst.In the process of 4 different experiments, by time difference microscopy observation altogether 100 embryos until the 5th day or the 6th day.Show the number percent of the cell that reaches each stage of indicating (8 cells, 4 to 7 cells, 2 to 3 cells and 1 cell).

Fig. 4 is a series of three the different embryos (top, middle part and bottom arrow) that pay close attention to for the period of indicating.

Fig. 5 is the chart showing for the time difference between the stage of this assessment, comprise the duration of cytokinesis for the first time, the time (being measured as the time interval between the end of cytokinesis 1 and the beginning of cytokinesis 2) for the first time and between second division and for the second time and for the third time time between mitosis (being measured as the time interval between initial sum cytokinesis 3 initial of cytokinesis 2).

Fig. 6 is the 3-D point diagram that shows the measurement of three events of a large group embryo, comprise the duration of cytokinesis for the first time, for the first time and for the second time the time interval between cell division (being measured as the time interval between the end of cytokinesis 1 and the beginning of cytokinesis 2) and cell and for the third time time interval between cell division (being measured as the time interval between initial sum cytokinesis 3 initial of cytokinesis 2) for the second time.The embryo's (by circular ring marks) who reaches the blastocyst stage is presented on 3-D figure and flocks together, and disperses everywhere reaching the embryo's (using X mark) who stagnates before blastocyst.

Fig. 7 shows that the recipient for using 3 dimension Morphologic Parameters prediction blastocysts to form operates the chart of sign (ROC) curve.

Fig. 8 is the radar chart showing from the gene expression dose of 1 to 2 cell stage of 6 stagnations and 52 genes of 5 normal 1 to 2 cell stages.As determined by graceful-Whitney inspection, the difference on the expression between normal and undesired embryo for highlight with yellow and with those genes of asterisk instruction be statistically significant.

Fig. 9 shows the histogram of heterogeneic expression in 2 cell stages stagnated and normal 2 cell stages.Top shows the time difference map picture of the selection quantity of 2 cell stages of stagnating.

Figure 10 is the histogram that shows the comparison of the homologous genes existing in Fig. 9 in 4 cell stages of stagnating and normal 4 cell stages.Top shows the time difference map picture of the selection quantity of 4 cell stages of stagnating.

Figure 11 is a series of histograms that show the gene expression pattern (ESSP) with 4 different modes.What indicate is the opportunity that the typical case of embryonic gene activation (the 2nd day) and the 3rd day expresses previous early stage transfer.

Figure 12 shows the gene expression from the gene of the single blastomere of different phase.(A) from two gene C TNNB1 and the gene expression of CDX2 and the variation on different phase for example 2 cells, 3 cells, mulberry body and these gene expression doses of blastocyst of marking and drawing at the single blastomere in different cell period.(B) gene expression in the parent program of the gene expression mark of column representative and the gene expression comparison from zygote program.

Figure 13 is for using time difference graphical analysis and relevant analysis of molecules to measure the figure of the model of embryo's viablity.

Figure 14 is a series of photos that show the three phases of growing in external Oocyte Maturation Process.

Figure 15 is a series of photos of the process of embryonic development after the external oocyte maturation of demonstration.

Figure 16 is the process flow diagram that shows the program for measuring egg mother cell.

Figure 17 is the process flow diagram that shows the program for measuring stem cell and pluripotent stem cell.

Figure 18 shows that the pluripotent stem cell of induction is divided into a series of photos of the process of neuron rose-shaped bunch (rosette).

Figure 19 is the table of measuring the classification that the gene of expression can be classified, comprises the number of every classification gene.

Figure 20 be four special patterns embryonic period, embryonic phase (ESSP) of determining in the embryo of 141 normal developments and the gene expression analysis of single blastomere and by described gene Clustering to each the table in these classifications.

Figure 21 shows the automated image analysis of the ability that proves the formation of imaging parameters prediction blastocyst.(A) show the result of single embryo's track algorithm.(B) show one group of analyzed 14 embryo.(C) show duration to cytokinesis and for the first time and the manual image analysis of the time between mitosis and the comparison of automatic analysis for the second time.(D) show good blastocyst morphology and the morphologic comparison of bad blastocyst.

Figure 22 is the synoptic diagram according to dark-field microscope of the present invention; The illustration on the left side shows the dark field sheet devices of Laser Processing.

Figure 23 is as the photo of three microscopical arrays described in Figure 22, is loaded on support and is connected to be arranged in couveuse and use with computer.Figure 23 A shows microscope and the inner couveuse of Figure 23 B demonstration microscope.

Figure 24 is the screenshot capture of the image capture software of use in this operation, shows the embryo from 3 passage imagings.

Figure 25 A to D shows from experiment 2, a series of four photos of the selected time difference map picture of position 2.Figure 25 A and 25B are the images of catching and Figure 25 C and 25D are the images of catching after nutrient culture media is changed before nutrient culture media is changed.

Figure 26 A and B are chart (left side) and the photos (right side) that shows normal and undesired embryo's cytoactive.Figure 26 A and 26B be presented at together that the 3rd day embryo has similar morphology but their cytoactive curve map but completely different and they in only a growth be blastocyst.

Figure 27 A and B are the figure with the customization Petri dish of micropore.Figure 27 A shows to have the figure of double dish of different dimensions and Figure 27 B shows the 3-D view of micropore.

Figure 28 A and B are the charts that shows the cytoactive and do not have with image registration in early stage.Figure 28 A shows that together with 28B registration has purified result and removed the peak owing to embryo's displacement or rotation.

Figure 29 A and B are chart (left side) and the cell photos (right side) that shows normal and undesired embryo's cytoactive.Figure 29 A and Figure 29 B be presented at together that the 3rd day embryo has similar morphology but their cytoactive curve map but completely different and they in only a growth be blastocyst.

Figure 30 is the chart that shows the difference of the pixel intensity between continuous pairs image during embryonic development.This can be used using mensuration embryo's viablity or as using how many particles (embryo model of prediction) to improve the method for for example particle filter of other algorithms by determining by independent.

Figure 31 A-G is a series of seven photos that show the result of following the tracks of from 2D of different cell stages.Cell processes is by indicating the frame number being associated with each photo: 15 frames (Figure 31 A), 45 (B), 48 (C), 189 (D), 190 (E), 196 (F) and 234 (G).Bottom arrow shows the analog image covering.Profile is visible cell membrane and white dashed line is inaccessible film.Within every 5 minutes, catch picture frame and only shown some.

Figure 32 A and B are a series of photos and the figure that shows two examples of many successful of 3D tracking cell.Explanation under every photo of embryo shows the vertical view of 3D model, and except frame 314 and frame 228, it is the side view of display model in frame 314 and frame 228 respectively.Within every 5 minutes, catch picture frame.

Figure 33 is the graphic representation of the fissional particle filter result of 1 cell to 2.Data point is the 3D location of cell centre.Point shows 1 cell model, 2 cell models, 3 cell models and 4 cell models.Top arrow shows the particle after prediction and the particle after bottom arrow demonstration resampling.

Figure 34 A and B are the charts that shows the contrast of the automatic and manual analyzing to one group of 14 embryo.Figure 34 A shows the contrast of duration of cytokinesis for the first time and Figure 34 B shows for the first time and the contrast of the time between mitosis for the second time.

Figure 35 shows how graphical analysis is used to model embryo and measures the process flow diagram of some Morphologic Parameters.

Detailed Description Of The Invention

Before describing this method and composition, should be understood that, the invention is not restricted to described concrete grammar and composition, because these can change certainly.Scope of the present invention it is to be further understood that at term used herein only for describing the object of particular, and is unexpectedly restrictive, because will be only defined by the appended claims

In the time that the scope of numerical value is provided, should be understood that, the each intermediate value (certainly 1/10th of the unit to described lower limit, except context otherwise clearly states) between the upper and lower bound of described scope is also by open especially.Each less scope in the value of any appointment in the scope of specifying or intermediate value and the scope in this appointment between value or the intermediate value of any other appointment is contained in scope of the present invention.The upper and lower bound of these less scopes can comprise independently or get rid of within described less scope, each scope arbitrary, described in being included in without one or two limit values within less scope time also comprises in scope of the present invention, and is subject to the special restriction of getting rid of any value in specified scope.In the time that the scope of specifying comprises one of two limit values or both, get rid of within one of included limit value or both scopes be also included within scope of the present invention.

Except defining in addition, all technical terms used herein are identical with common the understood implication of scientific terminology and the technical field of the invention those of ordinary skill.Can be for practice of the present invention or test at those any method and material described herein although be similar to or be equal to, some possible and preferred method and materials have been described now.All publications of mentioning are in this article incorporated to relevant method and/or the material quoted to described publication with disclosure and description by reference herein.Should be understood that, in the time there is contradiction, present disclosure substitutes any open of the publication that is incorporated to.

Should be noted that, as herein and use in subsidiary claim, otherwise clearly stating in context, singulative " (a) ", " one (an) " and " being somebody's turn to do (the) ", comprise plural referent.Therefore, for example, mention that " cell " comprises multiple such cells, mention that " this peptide " comprises to mention one or more peptides and its equivalent well known by persons skilled in the art, for example polypeptide, etc.

The publication of discussing is herein only for its openly providing before the application submits day to.Should not be construed as herein and admit that the present invention does not have right to rely on formerly invention and prior to above-mentioned publication.In addition, the date of the publication providing may be different from the actual publication date, and it may need to be confirmed separately.

definition

Method, composition and kit for determining one or more embryos or the developmental potentiality of multipotential cell and/or the existence of one or more embryo or multipotential cell chromosome abnormality are provided.These methods, composition and kit are determined in embryo the most useful in the sterility in the treatment mankind and egg mother cell useful in vitro.These and other object, advantage and feature of the present invention becomes apparent concerning those skilled in the art in the time of the details of the subject methods of reading as below describe more all sidedly and composition.

Term " developmental potentiality " and " growth competence " are used to mean ability (ability) or the ability (capacity) of healthy embryo or multipotential cell growth or growth herein.

Term " embryo " be used to herein to mean when for example unfertilized secondary oocyte of two haploid gamete cells and sperm in conjunction with form zygote that dliploid totipotent cell for example forms when zygote and by immediately subsequently until the embryo of mulberry body 16 cell stages and for example embryo's spilting of an egg of the cell division in blastocyst stage (trophectoderm of differentiation and inner cell mass) generation.

Term " multipotential cell " is used to mean to have any cell of the ability that is divided into polytype cell in biosome herein.The example of multipotential cell comprises stem cell egg mother cell and 1 cell stage (being zygote).

Term " stem cell " is used to mean cell or cell mass herein, its: the ability (a) with self and (b) have the potentiality of the cell type that produces different differentiation.Conventionally, stem cell has the potentiality of the cell that produces multiple pedigree.As used herein, stem cell can be myeloid-lymphoid stem cell, for example egg mother cell of fertilization, and it produces all embryos and the embryo outside organization of biosome; Pluripotent stem cell, dry (ES) cell of for example embryo, dry (iPS) cell of pluripotency of embryo's embryo (EG) cell or induction, it produces all embryonic tissues of biosome, i.e. entoderm, mesoderm and ectoderm pedigree; Multipotential stem cell, for example interstital stem cell, it produces at least two kinds in the embryonic tissue of biosome,, in entoderm, mesoderm and ectoderm pedigree at least two kinds or its can be tissue specificity stem cells, and it produces polytype noble cells of particular organization.Tissue specificity stem cell comprises the tissue specificity embryonic cell of the cell that produces particular organization and is arranged in adult tissue and can produces the soma cell of the cell of this tissue, for example produce all cells of central nervous system neural stem cell, produce the satellite cell of skeletal muscle and produce the candidate stem cell of all cells of hemopoietic system.

Term " egg mother cell " is used to mean unfertilized female egg mother cell or gamete herein.The application's egg mother cell can be first oocyte, in this case they be placed by or by meiosis I, or secondary oocyte, they are placed by or are passing through meiosis II in this case.

" meiosis " means to cause gametogenic cell cycle events.In initial meiosis cell cycle or meiosis I, the chromosome of cell is replicated and divides to two daughter cells.These daughter cells separate not following in synthetic second meiotic division cell cycle of DNA or meiosis II afterwards, produce the chromosomal gamete with haploid number.

" germ-vesicle " stage means the ripe stage of the first oocyte relevant with the I in early stage of meiosis I cell cycle, before the first division of caryoplasm.The egg mother cell in this period is also referred to as " germ-vesicle egg mother cell ", because giant cell core is peculiarly called germ-vesicle.In the normal human oocytes of cultivating in vitro, germ-vesicle occurs for about 6-24 hour after maturation starts.

" I in the mid-term " stage means the ripe stage of the first oocyte relevant with the I in mid-term of meiosis I cell cycle.Compared with germ-vesicle egg mother cell, mid-term I egg mother cell do not have large, the clear nucleus defining.In the normal human oocytes of cultivating in vitro, mid-term, I occurred for about 12-36 hour after maturation starts.

" II in the mid-term " stage means the ripe stage of the secondary oocyte relevant with the II in mid-term of meiosis II cell cycle.Mid-term, II can distinguish by the discharge of first polar body.In the normal human oocytes of cultivating in vitro, mid-term, II occurred for about 24-48 hour after maturation starts.

What " mitotic cell cycle " meant to cause in cell cell chromosomally copies and the tenuigenin of these chromosomes and cell is divided to the event in two daughter cells.The mitotic cell cycle is divided into two periods: interkinesis and mitosis.In the interkinesis, Growth of Cells and copy its DNA.In mitosis, cell is initial and complete cell division, first separates its caryoplasm and afterwards the caryoplasm (cytokinesis) of its kytoplasm and its separation is assigned in two cells that separate.

Egg mother cell that " mitotic cell cycle for the first time " or " cell cycle 1 " mean to be fertilized from fertilization to cytokinesis event is for the first time split into the time interval of two daughter cells.Egg mother cell by example in vitro fertilization in, the time interval of injection human chorionic gonadotrophin (HCG) (conventionally using before egg mother cell extracts) to the time of cytokinesis for the first time between completing can be used as the alternative time interval.

The cell cycle events for the second time that " mitotic cell cycle for the second time " or " cell cycle 2 " mean to observe in embryo, produce daughter cell and the time interval between first group of third generation cell of generation (granddaughter cell) of from these daughter cells (" conductor cell " or daughter cell A) by mitosis by mitosis from the egg mother cell of fertilization, in the time completing cell cycle 2, embryo is made up of 3 cells.In other words, the time between cell cycle 2 embryo that can be defined as intuitively the embryo who comprises 2 cells and comprise 3 cells.

The cell cycle events for the third time that " mitotic cell cycle for the third time " or " cell cycle 3 " mean to observe in embryo, normally produce daughter cell to from the second daughter cell (" delay daughter cell " or daughter cell B) produces second group of third generation cell time interval by mitosis by mitosis from the egg mother cell of fertilization, in the time completing cell cycle 3, embryo is made up of 4 cells.In other words, the time between cell cycle 3 embryo that can be defined as intuitively the embryo who comprises 3 cells and comprise 4 cells.

" First cleavage event " means first division, that is, egg mother cell is split into two daughter cells, i.e. cell cycle 1.In the time that First cleavage event completes, embryo is made up of 2 cells.

" spilting of an egg event for the second time " means second component and splits, that is, conductor cell division is that two third generation daughter cells and delay daughter cell are split into two third generation daughter cells.In other words, by cell cycle 2 and cell cycle 3, both form spilting of an egg event for the second time.In the time that spilting of an egg event for the second time completes, embryo is made up of 4 cells.

" spilting of an egg event for the third time " means the 3rd component and splits, that is, and and the division of all third generation daughter cells.In the time that spilting of an egg event for the third time completes, embryo is made up of 8 cells.

" cytokinesis " or " cell division " means cell and experiences fissional mitotic period.In other words, its be the caryoplasm of separation of cell and its kytoplasm by separately to produce the mitotic stage of two daughter cells.The time period of cytokinesis can be confirmed as when the contraction (" cleavage groove ") of cell membrane by first observed then and i.e. time period or the windows of two daughter cells between producing of the end of this contraction event.The initial curvature that can be defined as intuitively cell membrane of cleavage groove becomes the point of recessed (turning-in has indenture or recessed) from protruding (outside is circular).This is by o'clock being described in Fig. 4 top group in the white arrow at 2 cleavage groove places.Cell elongation initial can be used for the initial of mark cytokinesis equally, and the time period of cytokinesis is defined as the time period between the fissional end of initial sum of cell elongation in this case.

" cytokinesis for the first time " or " cytokinesis 1 " means the cell division event for the first time after fertilization, and the egg mother cell of fertilization divides to produce two daughter cells.Cell division for the first time usually occurs in fertilization one day after.

The cell division event for the second time that " cytokinesis for the second time " or " cytokinesis 2 " means to observe in embryo, i.e. a daughter cell (" conductor cell " or filial generation A) of the egg mother cell of fertilization division is split into first group of two third generation.

The cell division event for the third time that " cytokinesis for the third time " or " cytokinesis 3 " means to observe in embryo, i.e. another daughter cell (" delay daughter cell " or filial generation B) of the egg mother cell of fertilization division is split into second group of two third generation.

The article that term " base beacon note (fiduciary marker) " or " reference mark " are used in the field, the visual field of imaging system, it appears in produced image, is used as point or the tolerance of reference.It can be placed in imaging object or on something or or a group echo thing in the cross curve of optical instrument.

Term " micropore " refers to the container of cellular level size, is preferably provided for receiving single eukaryotic.

interested multipotential cell and embryo

In the method for the invention, by measuring one or more cell parameterses of one or more embryos or multipotential cell and these being measured to the developmental potentiality of measuring described embryo or multipotential cell for the developmental potentiality of definite described embryo or multipotential cell.Consequent information can be used to instruct clinical decision, for example, whether shift the embryo of fertilization, whether implants one or more cells of cultivation.

The embryo's that can measure by method of the present invention example comprises 1 cell stage (also referred to as zygote), 2 cell stages, 3 cell stages, 4 cell stages, 5 cell stages, 6 cell stages, 8 cell stages etc., conventionally up to 16 cell stages and comprise 16 cell stages, wherein any can by any mode easily from for example the egg mother cell of cylinder mature or the egg mother cell of maturation in vitro produce.

The example of the multipotential cell that can measure by method of the present invention comprise myeloid-lymphoid stem cell for example egg mother cell such as first oocyte and secondary oocyte; Pluripotent stem cell is ES cell, EG cell, iPS cell and similar cell for example; Pluripotent cell, for example interstital stem cell and tissue specificity stem cell.They can be from any stage of life, for example embryo, neonatal, juvenile, grow up with any sex, i.e. XX or XY.

Embryo and multipotential cell can derive from any biosome, such as such as the mankind, primate, equine species, bovid, porcine animals, canid, cats etc. of any mammalian cell kind.Preferably, they derive from the mankind.They can be frozen before, for example, and the freezing preservation of 1 cell stage and the embryo of thawing afterwards or egg mother cell and stem cell freezing and that thaw.Selectively, they can be fresh preparations, for example, and the embryo by technology in vitro fertilization from the fresh preparation of egg mother cell; By the fresh collection of maturation in vitro technology and/or fresh maturation or derive from the egg mother cell that vitro differentiation is reproduction cell and the ripe pluripotent stem cell for egg mother cell; The dissociating and cultivating stem cell and the similar cell of fresh preparation from tissue by methods known in the art.Under any applicable condition that they can be known in the art, cultivate to promote to need survival, growth and/or the growth of determined sample, for example, for embryo, in field in vitro fertilization for example under the condition of used those; Referring to for example United States Patent (USP) the 6th, 610, No. 543, United States Patent (USP) the 6th, 130, No. 086, United States Patent (USP) the 5th, 837, No. 543, its disclosure is incorporated to herein by reference; For egg mother cell, used to promote under those condition of oocyte maturation in this area for example; Referring to for example United States Patent (USP) the 5th, 882, No. 928 and United States Patent (USP) the 6th, 281, No. 013, its disclosure is incorporated to herein by reference; For stem cell, used to promote under those condition of propagation in this area for example; Referring to for example United States Patent (USP) the 6th, 777, No. 233, No. 7037892nd, United States Patent (USP), United States Patent (USP) the 7th, 029, No. 913, United States Patent (USP) the 5th, 843, No. 780 and United States Patent (USP) the 6th, 200, No. 806, No. 2009/0047263rd, U.S. Patent application, No. 2009/0068742nd, U.S. Patent application, its disclosure is incorporated to herein by reference.In the commercial available nutrient culture media that need to supplement serum or serum material, amino acid and growth factor for example KnockOut DMEM, the DMEM-F12 of specific embryo/multipotential cell that conventionally, embryo/multipotential cell is measured in cooperation or Iscoves improvement Dole Becquerel nutrient culture media, cultivate.

time difference imaging analysis

In some embodiments, embryo/multipotential cell is by measuring via time difference imaging measurement cell parameters.Embryo/multipotential cell can be cultivated in standard culture.Selectively embryo/multipotential cell can be cultivated in customization double dish for example has the customization double dish of the micropore of optical quality as described herein.In these customization double dish, the lower surface that each micropore is equipped with a single embryo/multipotential cell and each micropore has single small-sized microscope imaging simultaneously that whole group of embryo in optical quality smooth finish so that single ware can be by having enough resolution to follow cell mitogen process.The micropore of whole group share same nutrient culture media water dropper (drop) in double dish and can comprise be placed on micropore around for stablize the external holes of nutrient culture media water dropper and the fiducial marker of contiguous micropore placement.Available plasma etching or another kind of processing are adjusted surperficial hydrophobicity and in micropore, are formed bubble when preventing from being full of with nutrient culture media.No matter use standard culture still to customize double dish, between culture period, can in same nutrient culture media, cultivate the embryo of one or more growths, for example every ware can be cultivated the embryo between 1 to 30.

Obtain in time image and afterwards analysis image to obtain the measurement of one or more cell parameterses.The customization mini microscope array that available assembling is for example assembled the inverted microscope of warm table and incubator or is suitable for conventional couveuse inside for any computer-controlled microscope of digital picture storage and analysis carries out time difference imaging.The array of mini microscope make the sample of the multiple wares in same couveuse can cultivate simultaneously and size adjustable to adapt to multiple passages, the minimum interval between consecutive image is caught does not limit.Use multiple microscopes to eliminate the needs of mobile example, it has increased the reliability of system accuracy and whole system.Single microscope in couveuse can be partly or entirely to separate, for each double dish provides the environment of the control of self.The environment that this allows ware to be transferred to image space or not affect other samples from image space transfer.

Can use the illumination of the bright visual field, dark field illumination, phase correlation, Huffman modulation phase contrast (Hoffman modulation contrast), elementary errors to interfere contrast or fluorescence for the imaging system of time difference imaging.In some embodiments, dark field illumination can be utilized for feature extraction subsequently and graphical analysis provides the image comparison of enhancing.In addition, redness or near-infrared light source can be used to reduce phototoxicity and improve the contrast between cell membrane and the interior section of cell.

During the image obtaining can be stored in motion video in continuous substrate or be interrupted in substrate (as in time difference photography), wherein object is imaged on static photo repeatedly.Preferably, the time interval between image should be to catch as described below important morphology event between 1 to 30 minute.In selectable embodiment, the time interval between image can be depending on the amount of cytoactive and changes.For example, during activity, can every several seconds or per minute obtain so continually image, and during nonactive, can every 10 or 15 minutes or obtain more for a long time image.Can be used to when and how transformation period interval of detection to the realtime graphic analysis of caught image.In our method, the total amount of the light of sample reception estimates to equal imaging in the 5 days continuous low-level exposure of about 24 minutes.Be generally used for the microscopical light intensity of supplementary reproduction for the light strength ratio of time difference imaging system much lower, for example, owing to the low-power (using the 1W LED than common 100W halogen bulb) of LED and the high sensitive of camera sensor.Therefore the amount that, uses the energy that the total amount of the luminous energy that received by embryo of time difference imaging system receives during can the routine operation clinical with IVF almost or still less.In addition, the time shutter can significantly shorten to reduce the total amount of the exposure to embryo/multipotential cell.For the imaging of 2 days, at every 5 minutes, with 0.5 second exposure/image capture image in the situation that, the total amount of low-level exposure was less than 5 minutes.

After image acquisition, extract and for example size of the different cell parameters of analysis image, the thickness of oolemma, broken degree, the symmetry of the daughter cell producing from cell division, duration of the time interval between mitosis and cytokinesis several times.

Can pass through the normally morphology event of cell parameters of time difference imaging measurement.For example, in the time measuring embryo, time difference imaging can be used to measure the duration of for example cytokinesis 1 of cytokinesis event, cytokinesis 2, cytokinesis 3, cytokinesis 4, and the duration of wherein cytokinesis event is defined as first observed and is separated into the time interval between two daughter cells (i.e. the generation of two daughter cells) to cleavage groove (cytokinesis initial) and cleavage groove.The duration of interested another parameter is cell cycle events for example cell cycle 1, cell cycle 2, cell cycle 3 or cell cycle 4, wherein the duration of cell cycle events be defined as a cell (for the cell cycle 1, the fertilization of ovum; For after cell cycle, in the time that cytokinesis finishes) generation and from the time interval between the generation of two daughter cells of this cell.Can comprise the time interval by these cell event definitions by interested other cell parameterses of time difference imaging measurement, in for example time interval between (a) cytokinesis 1 and cytokinesis 2, may be defined as any of interval between the end of interval between the end of initial sum cytokinesis 2 of interval between interval, the end of cytokinesis 1 and the end of cytokinesis 2 between initial sum cytokinesis 2 initial of cytokinesis 1, cytokinesis 1 or cytokinesis 1 and cytokinesis 2 initial; Or in (b) time interval between cytokinesis 2 and cytokinesis 3, may be defined as any of interval between the end of interval between the end of initial sum cytokinesis 3 of the interval between initial sum cytokinesis 3 initial of cytokinesis 2 or the interval between the end of cytokinesis 2 and the end of cytokinesis 3 or cytokinesis 2 or cytokinesis 2 and cytokinesis 3 initial.

For object in vitro fertilization, be for example considered in favourable growth in early days the 2nd day or the 3rd day until 8 cell stages are transferred to uterus to reduce embryo's loss (shortcoming owing to condition of culture with respect to external environment) and to reduce the possible adverse consequences relevant with the rear raw mistake that can occur in the training period (people (2009) Hum Mol Genet.18 (20): the 3769-78 such as Katari by embryo; People (2009) Fertil Steril.91 (5): the 1765-70 such as Sep ú lveda).Therefore, preferably, although the time of analyzing is longer, for example approximately 36 hours, approximately 54 hours, approximately 60 hours, approximately 72 hours, approximately 84 hours, approximately 96 hours or longer, the measurement of the cell parameters that the same expection of this method occurred in 2 days of fertilization.

The example of the cell parameters of ripe egg mother cell that can measure by time difference imaging includes but not limited to the morphologic variation of egg mother cell cell membrane, the speed and the degree that for example separate from oolemma; The morphologic variation of ovocyte karyon, for example germinal vesicle breakdown (GVBD) initial, complete and speed; The movement velocity of particle and direction in tenuigenin and nucleus; The motion of the cytokinesis of egg mother cell and first polar body and the discharge of first polar body and/or duration.Other parameter comprises the duration of the cytokinesis of ripe secondary oocyte and second polar body.

The example of the cell parameters in stem cell or the population of stem cells that can measure by time difference imaging includes but not limited to the duration of cytokinesis event, time between cytokinesis event, the size and shape of stem cell before cytokinesis event and in process, the number of the daughter cell being produced by cytokinesis event, the dimensional orientation of cleavage groove, the speed of viewed asymmetric division and/or number (being that one of them daughter cell keeps stem cell and other differentiation), the speed of viewed symmetrical division and/or the end of number (wherein two daughter cells all keep stem cell or all break ups) and cytokinesis event and the time interval between stem cell starts to break up.

Parameter can manual measurement or they can automatically be measured, for example pass through image analysis software.In the time using image analysis software, can use the image analysis algorithm of utilizing taking sequential Monte Carlo method as basic probabilistic model estimating techniques, for example produce the distribution of embryo/multipotential cell model of supposition, taking simple optical model as basic analog image and by these simulations and the view data comparison of observing.In the time using such probabilistic model estimation, cell can be modeled as any applicable shape, for example oval set in 2D space, ellipsoidal set and analogous shape in 3d space.Block and degree of depth uncertainty the practicable geometrical constraint corresponding to desired entity behavior of method in order to process.In order to improve robustness, can on one or more focal planes, catch image.

gene expression analysis

In some embodiments, by measuring determination of gene expression embryo or multipotential cell.In these embodiments, cell parameters is gene expression dose or gene expression profile.Measure the expression of one or more genes, obtain express spectra or express assessment, can for example, for example, be undertaken by transcribed nucleic acid thing (mRNA) the expression of nucleic acid spectrum of measuring interested one or more genes; Or the level of one or more different protein/polypeptides of expression product by being measured as interested one or more genes is that protein groups express spectra carries out.In other words, term " express spectra " and " express assessment " by wide in range use to comprise the gene expression profile of rna level or protein level.

In some embodiments, the expression of gene can be composed to estimate by obtaining expression of nucleic acid, wherein be determined amount or the level of one or more nucleic acid in sample, the transcribed nucleic acid thing of for example interested one or more genes.In these embodiments, determined is nucleic acid samples with the sample that produces express spectra.Nucleic acid samples comprises the different nucleic acid of multiple or a group, and it comprises the expressing information of the interested gene of just evaluated embryo or cell.Nucleic acid can comprise RNA or DNA nucleic acid, and such as mRNA, cRNA, cDNA etc. obtain host cell certainly or the expressing information of tissue as long as sample retains it.Sample can be many different modes preparation, as known in the art, for example, by from cell separation mRNA, the mRNA that wherein separated is as knownly in differential expression field being used to amplification, using with preparation cDNA, cRNA etc.Use standard program, sample can be prepared from individual cells, the multipotential cell of the nutrient culture media of for example interested multipotential cell or the individual cells (blastomere) from interested embryo; Or from several cells, a part for the nutrient culture media of for example multipotential cell or interested embryo's 2,3 or 4 or more blastomere.

Express spectra can use any conventional program to produce from original nucleic acid sample.Known when producing the multitude of different ways of express spectra, for example in the field of differential genes expression analysis, use those time, be the gene expression profile generating routine with array basis for generation of representativeness of express spectra and the program of suitability type.These application are hybridization assays, wherein use in the spectrum that needs to be produced and to show for needing determined or carrying out each the nucleic acid of " probe " nucleic acid of the gene of analysis of spectrum.In these are measured, first from a sample of the original nucleic acid sample preparation target nucleic acid measured, wherein, preparation can comprise and carrys out tagged target nucleic acid by for example a member of signal representation system of label.After target nucleic acid sample preparation, sample is contacted with array under hybridization conditions, thus and be connected between the target nucleic acid of probe sequence complementation of array surface and form compound.Qualitative or detect quantitatively the existence of hybridization complex afterwards.

Practicablely comprise and be described in United States Patent (USP) the 5th, 143,854 to be created in the specific hybridization technology of the express spectra using in subject methods; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028; 5,800, No. 992, its disclosure is incorporated to herein by reference; And WO 95/21265; WO 96/31622; WO97/10365; WO 97/27317; Technology in EP 373203 and EP 785280.In these methods, described abovely will comprise that expressing just determined phenotype for it determines that each the array of " probe " nucleic acid of probe of gene contacts with target nucleic acid.For example under stringent hybridization condition, contact in hybridization conditions, and remove afterwards unconjugated nucleic acid.As used herein, term " strict condition determination " refer to be suitable for producing have enough complementarity with combination for example surface conjunction of nucleic acid and solution phase nucleic acid that desired specificity level is provided in mensuration to and be unsuitable for forming the right condition of combination having between the binding members that is not enough to the specific complementarity that provides desired.Strict complementary condition is summation or the combination (all) of hybridization and cleaning condition.

The pattern of the nucleic acid of the hybridization obtaining provides about each the expressing information in the gene of having been surveyed by probe, wherein whether expressing information is to express and the mode of what horizontal expression typically with gene, wherein expressing information, being express spectra (for example, to transcribe group (transcriptosome) form), can be quantitative and qualitative analysis.

Selectively, can use for by the method on non-array the basis quantitative level of one or more nucleic acid of sample, the mensuration that comprises those bases, for example PCR (PCR) taking amplification program as basis, comprises quantitative PCR, reverse transcription PCR (RT-PCR), PCR in real time and similar approach.

In some embodiments, the expression of gene can be assessed by obtaining protein groups express spectra, wherein determines amount or the level of one or more protein/polypeptides in sample, for example, by the protein/polypeptide of interested gene code.In these embodiments, determined is protein sample to produce the sample of the express spectra using in described method.When express spectra is protein groups express spectra, in sample when the spectrum of one or more protein levels, can use any conventional program for assessment of protein level, wherein determine the level of one or more albumen in the sample of measuring.

Although the mensuration for the multitude of different ways of protein level is as known in the art, for measuring a kind of representativeness of protein level and the scheme of suitability type is ELISA.In ELISA and the mensuration with ELISA basis, for interested albumen, specific one or more antibody can be fixed on selected solid surface, preferably the hole of for example polystyrene microtiter plates of the surface of display protein compatibility.Clean with after removing the material of incomplete absorption, will measure plate hole and use non-specific " sealing " albumen of known antigens neutrality for test for example bovine serum albumin(BSA) of sample (BSA), casein or milk power solution coated.This allows the non-specific adsorption site on sealing fixed surface, reduces thus the background being caused by the non-specific binding of antigen on described surface.Clean with after removing the closed protein in conjunction with sealing, by fixing surface with need tested sample and contact under the condition that contributes to immune complex (antigen/antibody) formation.These conditions comprise and contribute to equally to help to reduce being for example dissolved in BSA in hydrochloride buffer salt solution (PBS)/tween or PBS/Triton-X 100 or bovine gamma globulin (BGG) dilute sample and allowing sample to hatch about 2-4 hour the temperature (although also can use other temperature) of approximately 25 DEG C-27 DEG C with dilution of non-specific background.After hatching, clean the surface of antiserum contact to remove nonimmune compound material.Exemplary cleaning procedure comprises with solution for example PBS/ tween, PBS/Triton-X 100 or borate buffer solution cleaning.Then the appearance that immune complex forms can be determined by the combination that makes the immune complex experience of combination have specific second antibody and detection second antibody to the target different from first antibody with amount.In certain embodiments, second antibody has the enzyme of institute's combination, for example urease, peroxidase or alkaline phosphatase enzyme, and it will produce coloured precipitation in the time hatching with suitable chromogenic substrate.For example, can use anti-IgG a period of time that urease or peroxidase yoke close and for example, under the condition that is conducive to the development that immune complex forms (solution that is containing PBS for example in PBS/ tween in incubated at room 2 hours).After hatching by second antibody like this and cleaning to remove unconjugated material, by quantitative the amount of mark, for example by the example of urease mark with for example urea of chromogenic substrate and bromcresol purple or in the example of peroxidase labelling thing with 2,2 '-Lian nitrogen-bis--(3-ethyl-benzothiazoline)-6-sulfonic acid (ABTS) and H ₂o ₂hatch.For example use visible spectrum spectrophotometer to obtain quantitatively by measuring the degree of color generation afterwards.

First form before can be by being combined sample to change with assay plate.Afterwards primary antibody and assay plate are hatched, use afterwards primary antibody primary antibody to the secondary antibodies detection combination of specific mark.

The solid matrix that one or more antibody are fixed thereon can by being permitted, multiple material be made and permitted various shape such as microtiter plate, microballon, dipstick, resin particle etc.Can select matrix to maximize signal to noise ratio (S/N ratio), thus minimum background in conjunction with and be convenient to separate and coated.The mode that can be applicable to used matrix is implemented to clean, and for example, by microballon or dipstick are removed from reservoir, for example micro titer plate well of reservoir is emptied or dilute or with cleaning solution or solvent rinsing pearl, particle, chromatographic column or filter.

Selectively, can use the method with non-ELISA basis of the level for measuring one or more albumen of sample.Representational example includes but not limited to mass spectrum, protein groups array, xMAPTM Microspheres Technique, flow cytometry, Western blotting and immunohistochemistry.

The result obtaining provides the information relevant with each expression in the gene of being surveyed by probe, wherein expressing information whether express with gene and conventionally with the mode of what horizontal expression and wherein expression data can be qualitative and quantitative.

In the time producing express spectra, in some embodiments, working sample comprises at least one genes/proteins, the express spectra of the expression data of multiple genes/proteins sometimes to produce, wherein plural number means at least two kinds of different genes/proteins and often at least about 3 kinds, conventionally at least about 10 kinds or more, usually at least about 15 kinds of different genes/proteins or more, for example 50 kinds or more or 100 kinds or more etc.

In broadest sense, it can be qualitative or quantitative expressing assessment.Like this, when detection is qualitatively time, whether described method provides reading or evaluation, for example, assess for example nucleic acid of target analyte or expression product and be present in determined sample.Also having in other embodiments, described method provides target analyte whether to be present in the quantitative detection in evaluated sample, estimates or for example nucleic acid in determined sample of assessment target analyte or substantial amount or the relative abundance of albumen.In these embodiments, if quantitatively detection can be that absolute or described method is while detecting the method for for example nucleic acid of two or more different analytes in sample or albumen to be relative.Like this, term " quantitatively " during for quantitative for example one or more nucleic acid of target analyte of sample or the context of albumen, can refer to definitely or relative quantification.Absolute quantitation can be by comprising concentration known one or more check analysis things and complete with reference to the detection level that is standardization target analyte with known check analysis thing (for example, by producing typical curve).Selectively, can by by the detection level between two or more different target analytes or amount relatively to provide each the relative quantification in two or more different analytes for example relative to each other to complete relative quantification.

The example of the gene of expression prediction zygote developmental potentiality comprises Cofillin (NM_005507), DIAPH1 (NM_001079812, NM_005219), ECT2 (NM_018098), MYLC2/MYL5 (NM_002477), DGCR8 (NM_022720), Dicer/DICER1 (NM_030621, NM_177438), TARBP2 (NM_004178, NM_134323, NM_134324), CPEB1 (NM_001079533, NM_001079534, NM_001079535, NM_030594), Symplekin/SYMPK (NM_004819), YBX2 (NM_015982), ZAR1 (NM_175619), CTNNB 1 (NM_001098209, NM_001098210, NM_001098210, NM_001904), DNMT3B (NM_006892, NM_175848, NM_175849, NM_175850), TERT (NM_198253, NM_198255), YY1 (NM_003403), IFGR2/IFNGR2 (NM_005534), BTF3 (NM_001037637, , and NELF (NM_001130969 NM_001207), NM_001130970, NM_001130971, NM_015537).Other genes that expression can be used as the cell parameters of prediction embryonic development potentiality are provided in Fig. 8.When obtaining gene expression dose and measuring, gene level is usually evaluated and to for example for example GAPDH of known gene constant in whole growth of standard control or RPLPO or at this time point, its expression of expressing in the sample of known gene carries out standardization afterwards.

Gene expression dose can be from individual cells, for example determine from interested embryo's blastomere or the egg mother cell of separation or from the cell of the separation of the nutrient culture media of stem cell etc., or they can be from such as interested embryo's of embryo 2,3 or 4 or more blastomere until and comprise interested whole embryo or from multiple cells of the nutrient culture media of stem cell until and comprise that the whole nutrient culture media etc. of stem cell determines.

Aspect other, the present invention includes for carry out Genotyping and the program of gene expression analysis on individual cells simultaneously.For embryo, this can be used for improving preimplantation genetic diagnosis (PGD), a kind of program of individual cells being removed and detected the caryotype defect of its DNA or the existence of specified disease gene from embryo.Our method allows heredity and gene expression analysis simultaneously.Described method comprises the following steps: that (1) collect individual cells in the nutrient culture media or damping fluid of small size, (2) use the potpourri of Genotyping and gene expression analysis primer to carry out a step reverse transcription and PCR (PCR) amplification, (3) cDNA that is less than the amplification of collecting equal portions after the PCR of 18 circulations is to maintain the linearity of amplification, (4) use for example quantitative PCR in real time of cDNA equal portions standard technique to carry out gene expression analysis, (5) using remaining sample to carry out second takes turns PCR and uses for example gel electrophoresis of standard technique to carry out Genotyping with further increase hereditary information and (6) of the object for Genotyping.

determine developmental potentiality from image and/or gene expression analysis

Once obtain cell parameters measurement result, described measurement is just used to determine the developmental potentiality of embryo/multipotential cell.As discussed above, term " developmental potentiality " and " growth sensitivity " refer to ability (ability) or the ability (capacity) of multipotential cell or tissue growth or growth.For example, in egg mother cell or embryo's example, developmental potentiality can be egg mother cell or embryo growth or grow ability (ability) or the ability (capacity) for healthy blastocyst.As another example, in the example of stem cell, developmental potentiality is growth or grows for interested one or more cells for example neuron, muscle, B or T cell and the cytoid ability of class (ability) or ability (capacity).In some embodiments, egg mother cell or embryo's developmental potentiality is that described egg mother cell or embryonic development are healthy blastocyst; Successfully to implant uterus; With process gestation and/or ability (ability) or ability (capacity) to live and to be born.In some embodiments, the developmental potentiality of multipotential cell is that described multipotential cell is grown ability (ability) or the ability (capacity) for promoting interested tissue in interested one or more cells for example neuron, muscle, B or T cell and similar cell and/or body.

" good developmental potentiality " means embryo/multipotential cell and statistically probably grows as expected, and it has 55%, 60%, 70%, 80%, 90%, 95% or the higher probability of growing as expected, for example 100% probability.In other words, proving once to obtain in 100 of definite cell parameters measurement result to good developmental potentiality in 55,100 in 60,100 in 70,100 in 80,100 in 90,100 in 95 or whole 100 100 embryos or multipotential cell in fact continues to grow really as expected.On the contrary, " bad developmental potentiality " means embryo/multipotential cell and statistically probably do not grow as expected, and it has as expected 50%, 40%, 30%, 20%, 10%, 5% or the probability still less of growing, for example 0% probability.In other words, only prove once to obtain in 100 of definite cell parameters measurement result to bad developmental potentiality 50,100 in 40,100 in 30,100 in 20,100 in 10,100 5 or embryo or multipotential cell still less in fact really continue as expected to grow.As used herein, " normally " or " healthy " embryo and multipotential cell prove good developmental potentiality and " abnormal " embryo and multipotential cell show bad developmental potentiality.

In some embodiments, cell parameters measurement result is used directly to determine the developmental potentiality of embryo or multipotential cell.In other words, the absolute value of measurement itself is enough to determine developmental potentiality.In the embodiment of use time difference imaging measurement cell odd number, such example includes but not limited to following, good developmental potentiality in its any instruction human embryos in alone or in combination: (a) for example continue about 0-30 minute, about 6-20 minute, the cytokinesis 1 of average about 12-14 minute; (b) cell cycle 1 of lasting about 20-27 hour for example about 25-27 hour; (c) about 8-15 hour, for example about 9-13 hour, has the time interval between the end of cytokinesis 1 and the beginning of cytokinesis 2 of mean value of about 11+/-2.1 hour; (d) about 0-5 hour, for example about 0-3 hour, has the time interval between initial sum cytokinesis 3 initial of the cytokinesis 2 of the averaging time of about 1+/-1.6 hour, i.e. synchronism.Alone or in combination, in any instruction human embryos, the example of direct measurement of bad developmental potentiality includes but not limited to (a) continue to exceed approximately 30 minutes for example approximately 32,35,40,45,50,55 or 60 minutes or more cytokinesis 1; (b) continue to exceed approximately 27 hours for example cell cycle 1 of 28,29 or 30 or more hours; (c) continue to exceed 15 hours for example 16,17,18,19 or 20 or more hours or be less than 8 hours for example approximately 7,5,4 or 3 or the end of cytokinesis 1 and the beginning of cytokinesis 2 still less hour between the time interval; (d) 6,7,8,9 or 10 or the initial sum cytokinesis 3 of the cytokinesis 2 of more hours initial between the time interval.

In some embodiments, by by its with from reference to or contrast the cell parameters measurement result comparison of embryo/multipotential cell and with this comparison result so that definite cell parameters measurement result that uses of the developmental potentiality to described embryo/multipotential cell to be provided.Term " reference " and " contrast " mean to be as used herein used to explain the cell parameters measurement result of given embryo/multipotential cell and provide standardized embryo or the cell determined to its developmental potentiality.With reference to or contrast can be known there is for example good developmental potentiality of desired phenotype and can be therefore positive with reference to or the embryo/multipotential cell of contrast embryo/multipotential cell.Selectively, described reference/contrast embryo/multipotential cell can be known do not there is desired phenotype and be therefore negative with reference to or the embryo/multipotential cell of contrast embryo/multipotential cell.

In certain embodiments, by obtained one or more cell parameters measurement results information relevant with the phenotype that compares the described embryo/cell to obtain and assess from comparable one or more cell parameters measurement results of single reference/contrast embryo/multipotential cell.Also having in other embodiment, by more deep information relevant from the phenotype that compares the described embryo/cell to obtain and assess from comparable one or more cell parameters measurement results of two or more different reference/contrast embryos or multipotential cell obtained one or more cell parameters measurement results.The cell parameters measurement result that for example, the described one or more embryos from assessed or multipotential cell can be obtained compares to obtain with described embryo/cell whether have the relevant definite information of interested phenotype with positive and negative embryo or multipotential cell.

As an example, the cytokinesis 1 that normal human subject embryo has good developmental potentiality is about 0-30 minute, about 6-20 minute more frequently, average about 12-14 minute, approximately 1,2,3,4 or 5 minute, approximately 6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 minutes is more frequently 21,22,23,24,25,26,27,28,29 or up to about 30 minutes in some instances.When indicating bad developmental potentiality with the longer time period that completes cytokinesis 1 from the normal embryo who assesses compared with viewed with reference to embryo.As second example, cell cycle 1 in normal human subject embryo i.e. the time from completed by precise and penetrating cytokinesis 1, conventionally in about 20-27 hour, more frequently at about 25-27 hour, approximately 15,16,17,18 or 19 hours, approximately 20,21,22,23 or 24 hours and in approximately 25,26 or 27 hours, complete more frequently more frequently.When indicating bad developmental potentiality with the cell cycle 1 longer from the normal embryo who assesses compared with viewed with reference to embryo.As the 3rd example, in normal human subject embryo the end of cytokinesis 1 and cytokinesis 2 to start be about 8-15 hour, more usually about 9-13 hour, has the mean value of about 11+/-2.1 hour, 6,7 or 8 hours, more frequently approximately 9,10,11,12,13,14 or up to about 15 hours.Indicate bad developmental potentiality with the cell cycle 2 long or shorter from the normal embryo who assesses compared with viewed with reference to embryo.As the 4th example, the time interval between the initial sum cytokinesis 3 of cytokinesis 2 in normal human subject embryo initial for the second time and for the third time mitotic synchronism be common about 0-5 hour, approximately 0,1,2 or 3 hour more frequently, there is the mean value of about 1+/-1.6 hour, indicate bad developmental potentiality with the longer interval completing between cytokinesis 3 of cytokinesis 2 from the normal embryo who assesses compared with viewed with reference to embryo.Finally, as can how to apply the example of this embodiment in the time using gene expression dose as the parameter of assessment developmental potentiality, Cofillin, DIAPH1, ECT2, MYLC2, DGCR8, Dicer, TARBP2, CPEB 1, Symplekin, YBX2, ZAR1, CTNNB1, DNMT3B, TERT, YY1, IFGR2, the lower expression of BTF3 and/or NELF with low 1.5-2 cell stages of assessing compared with normal reference 2 cell stages are viewed doubly, 2-doubly, 3-doubly, 4-doubly, 5-doubly, 10-doubly, 20-doubly, 50-doubly or 100-expression doubly indicate bad developmental potentiality, indicate good developmental potentiality and be equal to or greater than from the expression of the viewed expression of normal reference 2 cell stage.Other examples can come from empirical data, for example, need the one or more with reference to embryo or multipotential cell of determined embryo/multipotential cell side by observation.Can use anyly with reference to embryo/multipotential cell, what for example have a good developmental potentiality is normal with reference to sample or have the abnormal with reference to sample of bad developmental potentiality.In some instances, can use not only one with reference to sample, for example, normally with reference to sample with extremely both can use with reference to sample.

In some embodiments, use by time difference microscopy or by expression pattern analysis but be not that the cell parameters measurement result of obtaining by time difference microscopy and expression pattern analysis can be expected.In other embodiment, use the cell parameters measurement result of obtaining by time difference microscopy and the cell parameters measurement result of obtaining by expression pattern analysis to expect.

As discussed above, can measure and use one or more parameters to determine the developmental potentiality of embryo or multipotential cell.In some embodiments, the measurement of single parameter may be enough to obtain determining developmental potentiality.In some embodiments, use the measurement of not only parameter for example 2 cell parameterses, 3 cell parameterses or 4 or more cell parameterses to expect.

In certain embodiments, can expect for the mensuration of multiple parameters, because can provide higher susceptibility and specificity to the mensuration of multiple parameters.Susceptibility means to be correctly accredited as positive in fact positive ratio.This can be shown by alignment diagram:

Therefore, " positive " is to have good developmental potentiality to be about to grow for embryo's " feminine gender " of blastocyst has bad developmental potentiality to be about to can not to grow in the method for the embryo of blastocyst therein, and 100% susceptibility means test identification like this by all embryos that grow for blastocyst.In some embodiments, the susceptibility of mensuration can be approximately 70%, 80%, 90%, 95%, 98% or higher, for example 100%.Specificity means to be correctly accredited as negative negative ratio.This can be mathematically described as:

Therefore, the positive is to have good developmental potentiality to be about to grow for embryo's feminine gender of blastocyst is to have bad developmental potentiality to be about to can not to grow in the method for the embryo of blastocyst therein, and 100% specificity means test identification and will can not grow for blastocyst is by all embryos that stagnated before blastocyst stage.In some embodiments, the specificity of mensuration can be approximately 70%, 80%, 90%, 95%, 98% or higher, for example 100%.

As proved in below embodiment part and Fig. 7, the use of three parameters provides the specificity of 94% susceptibility and 93% and the cut off of 3 times of standard deviations that distribute to blastocyst.In other words, method of the present invention can correctly identify before the blastocyst stage that 94% chance (susceptibility) will bud into embryo's number and the embryo's that 93% chance (specificity) will be stagnated the number of blastocyst.In addition described specific mean value and/or cut off can be depending on data set for calculating these values and application-specific and revise.

In some embodiments, the assessment of embryo or multipotential cell comprises that generation comprises technician's assessment of curee's embryo/multipotential cell, for example written report of " developmental potentiality assessment ", " assessment of chromosome abnormality " etc.Therefore, subject methods can further comprise and producing or output provides the step of the report of the result of such assessment, described report can the form of electronic media (for example electronical display on computer monitor) or the form of tangible media (being for example printed on the report on paper or in other tangible media) provide.

As described herein, " report " is electronics or entity file.It comprises the report element of the interested information that provides relevant with the assessment of obtaining by method of the present invention.Can generate electronically wholly or in part curee's report.Curee report comprises the assessment of the possibility of the existence of assessment, the chromosome abnormality of the developmental potentiality of curee embryo at least or multipotential cell etc.Curee report can further comprise one or more in following: the 1) information relevant with proving installation; 2) ISP's information; 3) curee's data; 4) sample data; 5) detailed assessment report part, provides the information relevant with how obtaining assessment, the reference point that the cell parameters for example a) obtaining is measured, b) used, if any; With 6) other features.

Report can comprise the information about mechanism for testing, described information with carry out hospital that sample collection and/or data generate, clinical or laboratory is relevant.Sample collection can comprise how generating sample, for example, how gather in the crops sample and/or culture sample etc. how from curee.Data generate can comprise how obtaining image or analyzing gene express spectra how.This information can comprise with the Name & Location of such as mechanism for testing, carry out the laboratory technicians of described mensuration and/or input data identity, carry out and/or analyze position that date and time, sample and/or the result data measured store, measure in the Lot Number of reagent (such as kit etc.) of use and the relevant one or more details of similar information.The report field (Report field) with this information can be used the information being provided by user to fill conventionally.

Report can comprise the information about ISP, and described ISP can be positioned at the outer or healthcare facility of the healthcare facility at user place.The example of such information can comprise ISP's Name & Location, carries out the individual name that sample separation and/or data generate when consultant's title and needs or expectation.The report field with this information can be used the data stuffing of being inputted by user conventionally, and it can select (for example using drop-down menu) from prefabricated script is selected.In report other ISP's information can comprise with result about and/or with illustrative report the contact details of relevant technical information.

Report can comprise curee's data division, comprise from its collect egg mother cell or multipotential cell curee medical history, patient age, IVF cycles illness that has not attacked the vital organs of the human body (speed of being for example fertilized, the 3rd day follicular stimulating hormone (FSH) level) and when collect egg mother cell, zygote/embryo queue parameter (for example embryo's sum).The optimization number that these curee's data can be merged in to promote embryo's assessment and/or help to determine the embryo who shifts.Report also can comprise that for example definite curee's of managerial curee's data (namely nonessential information for assessment developmental potentiality) information is (such as name, curee's date of birth (DOB), sex, mailbox and/or inhabitation address, medical records number (MRN), room in medical and health organization and/or bed item), insurance information and similar information), arrange the responsible work doctor's (for example PCP) of curee's the doctor of assessment of developmental potentiality or other professional medical-care personnel's name and (if different from the doctor who arranges) curee's nursing name.

Report can comprise sample data part, its can provide with assess in the relevant information of biological sample analyzed, the type (type of embryo or multipotential cell and multipotential cell) of for example sample, how processing sample (for example storage temperature, preparation procedure) and the date and time collected.The report field with this information can be used the data stuffing of being inputted by user conventionally.Some of them can be provided as the selection (for example using drop-down menu) of prefabricated script.

Report can comprise assessment report part, and it can comprise assessing/determine relevant information with how obtaining as described in this article.Illustrative report can comprise the embryo that for example assessed or time difference map picture and/or the gene expression results of multipotential cell.The evaluation part of report also optionally comprises recommendation part.For example, in the time that result is indicated embryo's good developmental potentiality, recommendation can be included in the recommendation as a limited number of embryo implanted during the fertility treatment of this area recommendation to uterus.

What also should be readily appreciated that is that report can comprise other element or the element of modification.For example, in the time being electronics, report can comprise to point to provides the inside of more details or the hyperlink of external data base relevant with the element of selected report.For example, the patient information element of report can comprise that electronic patient records or obtain the hyperlink of the website of such patient's record, and described patient's record is retained in confidential data storehouse.This embodiment below may be interested in hospital system or clinical practice (clinic setting).In the time of form with electronics, report is recorded on applicable physical medium, such as, on computer-readable medium such as computer memory, zip driver, CD, DVD etc.

What should be readily appreciated that is that report can comprise all or some in element above, and condition is that described report generally includes the element that is at least enough to provide the needed analysis of user (assessment of for example developmental potentiality).

practical

As discussed above, method of the present invention can be used for assessing embryo or multipotential cell to determine their developmental potentiality.This of developmental potentiality determined and be can be used for instructing clinical decision and/or action.For example, in order to increase pregnancy rate, clinician is usually transferred to multiple embryos in patient, may cause all bringing the multifetation of health risk to mother and fetus.Use and obtain result from method of the present invention, can before implant, determine and will be transferred to grow the embryo's who is fetus developmental potentiality, allow doctor to determine to shift how many embryos to the opportunity of success of full-term pregnancy is maximized simultaneously by risk minimization.

The assessment of being undertaken by following method of the present invention also can be finding purposes by the embryo in one group of embryo or multipotential cell or multipotential cell according to their developmental potentiality classification.For example, in some instances, multiple embryos can grow for blastocyst, are about to have good developmental potentiality.But some embryo will more may reach blastocyst stage or the blastocyst higher than other quality, they will have the better developmental potentiality than other embryos.Under these circumstances, method of the present invention can be used to the embryo's classification in group.In such method, measure one or more cell parameterses of each embryo/multipotential cell to obtain the cell parameters measurement result of each embryo/multipotential cell.Be used to determine described embryo or multipotential cell developmental potentiality relative to each other from each the one or more cell parameters measurement results in described embryo or multipotential cell afterwards.In some embodiments, from described embryo or multipotential cell each cell parameters measurement result by by they directly be compared to each other to determine that the developmental potentiality of described embryo or multipotential cell uses.In certain embodiments, from described embryo or multipotential cell each cell parameters measurement result by by described cell parameters measurement result with from the cell parameters measurement result comparison with reference to embryo/multipotential cell with determine the developmental potentiality of each embryo or multipotential cell and afterwards definite developmental potentiality of more each embryo or multipotential cell use to determine described embryo or multipotential cell developmental potentiality relative to each other.By this way, for example multiple zygote/embryos' of assessment doctor can only select top-quality embryo, and those with best developmental potentiality shift to the opportunity of success of full-term pregnancy is maximized simultaneously by risk minimization.

The assessment of being undertaken by following method of the present invention also can be used for determining the developmental potentiality of the egg mother cell of maturation in vitro and the stem cell of in vitro culture.The information of the developmental potentiality about egg mother cell obtaining by method of the present invention can instruct the selection of doctor to the egg mother cell for being fertilized, and produces the higher successful possibility that obtains blastocyst from these egg mother cells.Equally, about the information of the developmental potentiality of stem cell can inform doctor to for example in its curee of needs in body restructuring or replace the program of tissue in the selection of the stem cell that uses.

reagent, equipment and kit

Provide equally one or more its reagent, equipment and the kit for carrying out above-described method.Its theme reagent, equipment and kit can alter a great deal.With regard to measuring any the method in cell parameters mentioned above, interested reagent and equipment comprise referred to above those, wherein these reagent can comprise array, antibody, signal production system reagent of culture plate, nutrient culture media, microscope, imaging software, imaging analysis software, nucleic acid primer, nucleic acid probe etc., depend on pending concrete process of measurement.For example, as described above, reagent can comprise that one or more in gene C ofillin, DIAPH1, ECT2, MYLC2/MYL5, DGCR8, Dicer/DICER1, TARBP2, CPEB1, Symplekin/SYMPK, YBX2, ZAR1, CTNNB1, DNMT3B, TERT, YY1, IFGR2/IFNGR2, BTF3 and NELF are had to specific PCR primer.Other examples of reagent comprise comprising that one or more in interested gene are had to specific primer or the array by the antibody of the albumen of interested these gene codes.

Except element above, theme kit is by the instructions further comprising for carrying out subject methods.These instructionss will be present in theme kit with various forms, and one or more can be present in kit.A kind of form that these instructionss can be present in is wherein the information as the medium applicable or suprabasil printing, and for example information is printed one or several sheets paper thereon, in the packaging of kit, medium at package insert.Also having another kind of mode will be that information has been recorded computer-readable medium thereon, such as floppy disk, CD etc.Can exist also have another kind of mode be can be via internet use the station address at mobile site acquired information.Any usual manner can be present in kit.

Use the automated cell imaging of array of microscopes

Some in above-described method need to be via the ability of time difference imaging embryo and development of stem cells.This can use by the system miniature, that hyperchannel array of microscopes forms that can be suitable for standard couveuse inside and obtain.This allows by multiple samples rapidly and imaging simultaneously and needn't mobile physically ware.A kind of illustrative prototype being shown in Figure 20 is made up of the 3 passage array of microscopes with dark field illumination, although can use the illumination of other types." triple channel " means three of the imagings simultaneously of three different double dish microscopes independently.The focal position that focuses on or obtain 3D rendering stack (image stack) with stepper motor adjustment.White light LEDs is used to illumination, although we have observed for human embryos redness or near infrared (IR) LED can improve the contrast between cell membrane and cell interior.The contrast of this raising can be assisted with manual and automatic imaging analysis.In addition, move to infrared region and can reduce the phototoxicity to sample.Obtain image by low cost high-resolution camera, but can use the camera of other types.

As shown in Figure 22, each microscope of above-described prototype system is used to comprise 1-30 the embryo's who does not wait double dish imaging.Microscope is collected the light from the white light LEDs being connected with the heat abstractor of any heat that helps dissipation to be produced by LED, and it is very little for the short time shutter.Light is by blocking the conventional dark field sheet of direct light, and " Petri dish " that arrives sample labeling by collector lens is upper, and it is the double dish that retains the embryo who is cultivated and study.Double dish can have and helps in the time ware being brought into or taken out of couveuse, to retain the order of cell and stop the hole that they move.Hole can be enough to approach together so that embryo can share same nutrient culture media water dropper on space.Scattered light is by micro objective, afterwards by achromatic doublet (achromat doublet) and to cmos sensor afterwards.Cmos sensor is connected so that graphical analysis as described above and spike as digital camera and with computing machine.

This design easily changes size much more passage and different lighting engineering to be provided and can be modified to adapt to the fluid device for sample introduction.In addition, design can be integrated with feedback control system, wherein by condition of culture for example temperature, CO ₂(to control pH) and nutrient culture media are based on feedback and from imaging data real-time optimization.This system is used to obtain the time difference video that human embryos are grown, and it has practicality in the embryo's viablity that is identified for (IVF) in vitro fertilization program.Other application comprise stem-cell therapy, drug screening and organizational project.

In an embodiment of equipment, illumination regulates and drives the Luxeon white light emission diode (LED) of energy supply to provide by being arranged on aluminium heat abstractor and by BuckPuck electric current.Light from LED passes through collimation lens.Afterwards as shown in Figure 22 collimated light by customization Laser Processing light barrier and use aspheric surface collector lens to be focused to the light cone of hollow.Directly transmission is refused by object lens by the light of sample, and is collected by the light of sample scattering.In one embodiment, use and there are the Olympus object lens of 20X magnification, although can use less magnification to increase the visual field, maybe can use larger magnification to increase resolution.The impact afterwards light of collecting being differed to reduce chromatin and sphere by achromatic doublet (being tube lens).Selectively, the light of collecting from image-forming objective lens can pass through finger conduct in the opposite direction another object lens that substitute to tube lens.In one embodiment, image-forming objective lens can be 10X object lens, and tube lens can be 4X object lens.The image obtaining can catch by the cmos sensor with 2,000,000 pixel resolutions (1600x1200 pixel).Also can use dissimilar sensor and resolution.

Figure 23 A shows the photo with 3 identical microscopical hyperchannel array of microscopes.All optical elements are installed in microscope tube.In the operation of array system, Petri dish is loaded on the polypropylene platform that is arranged on manual 2 axle tilting tables, and it allows with respect to optical axis adjustment as plane.These are fixed on microscopical base and after initial alignment and do not move.The light fixture being made up of LED, parallel light tube lens, light barrier and collector lens is mounted on manual xyz platform to place and focus illumination light.The image-forming assembly being made up of object lens, achromat and cmos sensor is arranged on manual xyz platform equally to the visual field is located and object lens are focused on.All 3 image-forming assemblies are connected to linear slide rail and are supported by the stepper motor driven single lever arm of use.This allow image stack computer control focusing and automatically catch.Can use the additive method of automatic focus and driving (actuation).

As shown in Figure 23 B, array of microscopes is placed on to standard couveuse inside.Cmos image sensor connects and is connected with the single hub (hub) that is positioned at couveuse inside through USB, and it is passed to the exterior PC of following other communication and power circuit.All electronic cable are drawn couveuse by the center of the rubber stopper with silicone adhesive sealing.

Above-described array of microscopes is used to record the time difference map picture of early stage human embryos growth and proved to start the growth through blastocyst stage from zygote.Four different experimental monitorings 242 embryos altogether.In this group, by 100 embryo's imagings until the 5th or 6 days; Other embryos are shifted out from image space in the different time points of gene expression analysis.The screenshot capture of image capture software and the embryo of imaging are shown in Figure 24.With every image roughly the low exposure of 1 second within every 5 minutes, catch image.The lasting exposure that the total amount of the light of sample reception equals 24 minutes, similar to the aggregate level experiencing in IVF clinic during processing.The exposure of 1 second duration of every image can reduce.Before with mankind's embryo operation, we have carried out many-sided control experiment with Preimplantation Embryos of Mouse and have not been subject to the impact of imaging process to guarantee blastocyst formation speed and gene expression pattern.

Figure 25 and 26 show from time difference sequence the image of selection.Show the image of the 1st day, the 2.5th day, the 4th day and the 5.5th day.For the sequence shown in Figure 25,3 growths in 9 embryos are blastocyst, and for the sequence showing in Figure 26,5 growths in 12 embryos are blastocyst.Pay close attention in time single embryo, even if their positions in photographed region are because embryo changes and shifts at the 3rd day experience nutrient culture media.Need to meet with Continuous Cultivation base the phase specificity demand of embryo in growth.Between the nutrient culture media stage of replacement, embryo is shifted out to a few minutes and is transferred to new Petri dish from image space.In order to grasp each embryo's identity between the nutrient culture media stage of replacement, sample is photographed to confirm that from a ware to the transfer of other wares embryo is not mixed up.Collecting for same this process that uses during the sample of gene expression analysis.The problem of following the trail of embryo's identity can be by being used hole with help, embryo to be arranged and solved with particular order.

There is the Petri dish of micropore

When shift Petri dish between different positions time, embryo may be shifted sometimes, causes and is difficult to grasp embryo's identity.Challenge has been proposed in the time carrying out time difference imaging a position and embryo is moved to second position for Embryo selection and transfer subsequently.A method is to cultivate embryo in independent Petri dish.But this needs each embryo to have its oneself nutrient culture media water dropper.In typical IVF program, be conventionally desirably in same Petri dish and with whole embryos of same nutrient culture media water dropper cultivation patient.In order to address this problem, we have designed the customization Petri dish with micropore.This prevents embryo's displacement and keeps their arrangements on Petri dish in the time transferring to or migrate out couveuse or imaging site.In addition, hole be enough little and space on be close together so that they can share same nutrient culture media water dropper and all being observed by same microscope simultaneously.The lower surface of each micropore has optical quality smooth finish.Figure 27 A shows the figure of the size with an embodiment.In this view, in the visual field of 1.7x1.7mm, there is the micropore being close together on 25 spaces.Figure 27 B shows the 3D view of micropore, in its recessed ware surface approximately 100 microns.On ware, comprise base beacon note (comprising letter, numeral and other marks), to help discriminating.

All lists of references of quoting are herein incorporated to herein with its entirety by reference.

Embodiment

List following examples how to carry out and to use disclosure of the present invention and description providing as those skilled in the art, and unexpectedly for restriction inventor thinks scope of the present invention, they do not mean yet and represent that following experiment is all or only experiment of carrying out.Endeavour to ensure the accuracy with regard to used numeral (as amount, temperature etc.), but should be taken into account some experimental errors and deviation.Except as otherwise noted, otherwise part be weight portion, molecular weight is weight average molecular weight, and temperature is degree Celsius that pressure is atmospheric pressure or approaches atmospheric pressure.

Sample source

In this research, all embryos used are that ability during is for many years collected and are fertilized and freezing preservation by multiple embryologists.In our research, each patient's average embryo number is 3 and is included in all age groups that general IVF center runs into.Notably, be that IVF produces (contrary with ICSI) for all embryos of these experiments, therefore embryonic origin is in the sperm (with regard to the ability that at least penetrates ovarian cumulus, oolemma and egg membrane and formation pronucleus with regard to them) with relatively normal function.Stimulation programs is the long lupron scheme (cdc.gov/art) of standard.Extra human embryonic freezing preservation completed by they are placed on to freezing nutrient culture media (1.5M 1,2 propylene glycol+0.2M sucrose) in room temperature (22+2 DEG C) upper 25 minute.Use afterwards (1 DEG C/min to-6.5 DEG C of slow freezing scheme; Keep 5 minutes; Inoculation; Keep 5 minutes;-0.5 DEG C/min to-80 DEG C; Drop in liquid nitrogen) by embryo cryopreservation.The council.Do not have shielded health and fitness information to be associated with embryo.

Confirm the embryo of the freezing preservation of a large group and carry out following observation: 1) embryo confirms the opportunity of the boundary mark mode of instruction normal embryo development, described boundary mark comprises: the spilting of an egg is 2 cells (the early stage generations at the 2nd day), the beginning (occurring at the 1st to 3 days) of RNA degraded, the spilting of an egg is 4 and 8 cells (occurring the 2nd day and the 3rd day late period respectively), the activation (at the 3rd day 8 cell stages) of embryonic gene group and the formation of mulberry body and blastocyst (respectively the 4th and occur for 5 days).2) embryo confirms the efficiency in the blastocyst stage that is reached for the embryo who typically obtains under clinical setting.This is probably frozen and represents this fact of array of the embryo who runs in the IVF consultation of doctors in the 2PN stage owing to embryo because be not carry out before the freezing preservation of the 1 cell stage embryo of the 3rd day or the more late freezing preservation of blastocyst stage (typically) will or such by not growing " class is selected method (triage) ".Therefore, our data acknowledgement with in typical IVF clinic, observe those compared with these embryos show similar blastocyst and form speed.3) embryo that the verified and fresh embryo of research before preserved than the 2PN stage shows the similar potentiality of growth, transplanting, clinical pregnancy and childbirth.Other research is for the similar result of the same demonstration of freezing egg mother cell, and our research shows equally, shows that for the similar result of freezing egg mother cell the event the earliest that human embryos are grown keeps reasonable time line after freezing preservation.4) we pay close attention to and do not depend on the time of fertilization or the parameter of thawing time.First parameter (cytokinesis for the first time duration) that we measure has the very short duration (approximately 10-15 minute) and does not depend on the time of the fertilization in this research (no matter its can by independent measurement and net result in all embryos).And, with respect to all parameters subsequently of this initial measurement point measurement and will compare successfully growing for blastocyst and fail to grow between the embryo for blastocyst.5) final, we notice that fresh (not freezing) embryo into 3PN is known and grow according to the time frame identical with fresh fetal tissues; We have compared the parameter the fresh 3PN embryo that we obtain from IVF clinic, Stamford and have observed them does not have different from those of the embryo of our freezing preservation or disclosed report.

Planning of experiments

In four experiments arrange, we follow the trail of the growth of 242 pronucleus phase embryos (being respectively 61,80,64 and 37).In each setting of experiment, at the 1st day, mankind's zygote thawed and cultivate on multiple plates with group.Under dark field illumination, use the each plate of time difference microscopy independent observation at different image spaces.With the time interval of about 24 hours, from imaging system remove the embryo of a plate and be collected as single embryo or single cell (blastomere) for high flux real-time quantitative PCR gene expression analysis.Each plate is generally comprised within whens results and reaches embryo's (being called " normally ") of desired stage of development and stagnate or postpone or the very big potpourri of those (being called " abnormal ") of fragmentation in the stage of development early.Using embryo as single complete embryo's analysis or be separated into single blastomere, carry out afterwards gene specific RNA amplification.By the embryo of a subgroup (in 242 100) imaging until the 5th or 6 days so that monitoring blastocyst forms.

Human embryos are cultivated and microscope

By freezing bottle is shifted out and they are positioned over to room temperature from liquid nitrogen storage, human embryos are thawed.Once bottle is thawed, open it and embryo visible under dissecting microscope.Afterwards the content of bottle is poured into the bottom of 3003 double dish, the survival that embryo is placed into water dropper and assessment and records each embryo.In room temperature, embryo is transferred to 3037 double dish, it comprises 1.0M 1,2 propylene glycol+0.2M sucrose 5 minutes, afterwards 0.5M 1,2 propylene glycol+0.2M sucrose 5 minutes and 0.0M 1,2 propylene glycol+0.2M sucrose 5 minutes.Subsequently, use the droplet under oil, between the 1st to 3 days, embryo is being supplemented to 10%Quinn ' s Advantage haemocyanin substitute (SPS; CooperSurgical) in Quinn ' s Advantage spilting of an egg nutrient culture media (CooperSurgical), cultivate and the 3rd day after, there is cultivation in Quinn ' the s Advantage blastocyst nutrient culture media (CooperSurgical) of 10%SPS.All these experiments are used the cleavage stage nutrient culture media of same type, and except two positions in first experiment, it uses Global nutrient culture media (LifeGlobal, Guilford, CT).In this little subgroup (12 embryos), embryo shows low a little blastocyst and forms speed (in 12 3, or 25%), but susceptibility and the specificity of organizing our Prediction Parameters for this are all 100%.

In multiple systems, carry out time difference imaging to adapt to analyze in multiple samples and the consistance of the data of checking different platform.System is made up of 7 different microscopes: (1) has equipped Tokai Hit warm table, white light Luxeon LED and the Olympus IX-70/71 microscope for two amendments of the aperture of dark field illumination; (2) equipped the Olympus IX-70/71 microscope (noting: determine that dark field illumination is only to use these systems during first in 4 experiments after preferred for measurement parameter) of two amendments of warm table, white light Luxeon LED and Huffman Modulation Contrast illumination (modulation phase contrast illumination); (3) be suitable for the customization 3 Channel Micro array of microscopes of standard couveuse inside, be equipped with white light Luxeon LED and the hole for dark field illumination.We observe in growth behavior, blastocyst forms between the embryo who cultivates in these different systems on speed or gene expression profile does not have marked difference; In fact, our parameter for blastocyst prediction is consistent between multiple systems and experiment.

The systematic light intensity of institute is all much lower than the normally used light of supplementary reproduction microscope, owing to the low-power (with respect to the halogen bulb of common 100W) of LED and the high sensitivity of camera sensor.Use light power meter, we determine that the power of the typical supplementary reproduction microscope of wavelength at 473nm (Olympus IX-71 Huffman modulation phase contrast) is in about scope of 7 to 10mW (depending on magnification), and the power of our imaging system identical wavelength measurement 0.2 and 0.3mW between.Caught image nearly 5 to 6 days with every 5 minutes of the time shutter of 1 second, produce the lasting exposure of approximately 24 minutes, at the power of 0.3mW, it equals under typical supplementary reproduction microscope the exposure of about 1 minute.

For the identity that the imaging relevant and gene expression experimental session are followed the trail of each embryo, we video camera has been installed on stereo microscope and record that nutrient culture media is changed and sample collection during the process of sample transfer.We carry out control experiment and do not observe significant difference (p=0.96) in the blastocyst formation speed between embryo imaging and contrast with the human embryos (n=22) of Preimplantation Embryos of Mouse (n=56) and a little subgroup.

High flux qRT-PCR analyzes

Analyze for single embryo or single blastocyst qRT-PCR, first with acid Tyrode solution-treated embryo to remove zona-free.In order to collect single blastomere, with accurate move liquid by embryo in 37 DEG C at Quinn ' the s Advantage with HEPES without Ca ²⁺mg ²⁺nutrient culture media (CooperSurgical) in cultivate 5 to 20 minutes.Sample is directly collected in 10 μ l reaction buffers, carries out as previously described subsequently a step reverse transcription/pre-amplified reaction.At reverse transcription with increase in advance between the reaction period the 20XABI assay-on-demand qRT-PCR primer merging and probe mixture (Applied Biosystems) as gene-specific primer.As previously described, carry out high flux qRT-PCR with the Fluidigm Biomark 96.96 Dynamic Array that use ABI assay-on-demand qRT-PCR probe.All samples repeats (technical replicate) with 3 or 4 technology and loads.Carry out qRT-PCR data analysis with qBasePlus (Biogazelle), Microsoft Excel and customized software.Some gene is omitted in data analysis, owing to the bad quality of data (for example bad pcr amplification curve) or in assessed embryo, be low to moderate all the time without express.For the blastomere analysis in period, the maternal transcript group using comprises DAZL, GDF3, IFITM1, STELLAR, SYCP3, VASA, GDF9, PDCD5, ZAR1 and ZP1, and embryonic gene group comprises ATF7IP, CCNA1, EIF1AX, EIF4A3, H2AFZ, HSP70.1, JARID1B, LSM3, PABPC1 and SERTAD1.Use geNorm and Δ Δ Ct method to calculate every kind of gene with respect to reference gene GAPDH and RPLPO and with respect to the expression value of gene mean value.In experience, based on gene stability value and the coefficient of variation (for GAPDH, being 1.18 and 46% and for RPLPO, is 1.18 and 34%), select GAPDH and the RPLPO reference gene as this research.These are the most stable and just in time in the scope of typical allos sample set in 10 house-keeping genes of our test.Second, we observe in single blastomere, as expected, the each division between 1 cell and 8 cell stages of the amount of GAPDH and RPLPO transcript reduces about 1Ct value, meets and before EGA, does not exist during the first three days of growing in the case of the mankind new transcript each cell along with the expection in the hereditary about half mRNA of each cleavage division pond.The 3rd, after we notice that EGA starts, in single blastomere, the expression of these reference genes is stable to maintenance between morula stage at 8 cells.In complete embryo's level, the Ct value of RPLPO and GAPDH until morula stage keeps constant to a great extent, has slight rising in blastocyst stage in whole growth, the transcript level increasing may exist owing to the blastomere of greater number time.The puberty of major part in the gene expression analysis carrying out in this research before concentrating on morula stage, in the time that the expression of reference gene is highly stable.

Automated cell is followed the tracks of

Our tracking cell algorithm uses taking sequential Monte-Carlo method as basic probabilistic framework, and it is often called as particle filter in computer vision field.Particle filter is followed the trail of the growth of three primary variabless in time: state, contrast and measurement.State variable is embryo's model and is expressed as oval set.Collative variables is the input of conversion conditions variable and is made up of our cell proliferation and branching model.Measurand is observation to state and is made up of our image obtaining by time difference microscopy.We distribute to represent by posterior probability to the estimation of the standing state in each time step, and it is similar to one group of weighting sample that is called particle.We can alternately use term particle and embryo mutually, and wherein particle is a hypothesis of embryo model preset time.After initialization, three steps of particle filter repeated application: prediction, measurement and renewal.

Prediction: oval and each cell that cell is represented as in 2D space has direction and overlapping index.Overlapping index describes the relative height of cell in detail.Conventionally, we wish to predict the behavior of two types: cell movement and cell division.For cell movement, our contrast input adopts each parameter of particle and the each cell of random permutation, comprises the length of position, direction and major axis and minor axis.Upset is randomly from having the normal distribution sampling of relatively little variance (initialized value 5%).For cell division, we use following method.Given point in time, for each particle, 50% the possibility that in our designated cell one will divide.This value is that experience is selected and comprises that possible cell division on a large scale keeps the good coverage of existing configuration simultaneously.If expected division, the cell of division will be selected at random so.When cell is selected to division, we use the symmetry division along oval major axis, produce two daughter cells of formed objects and shape.Each value of our random permutation daughter cell afterwards.Finally, we select the overlapping index of two daughter cells to keep their correct overlapping to the cell for remaining at random simultaneously.

After using contrast input, each particle is converted into analog image by we.This completes by using overlapping index the elliptical shape of each cell to be incident upon on the image of simulation.Corresponding pixel value is set to bi-values 1 and expands to produce the film thickness equal with viewed view data.Due to embryo be partially transparent and collect out of focus light, the cell membrane of embryo bottom is only sometimes visible.Correspondingly, there is 10% possibility and increase closed cell membrane.In fact, we have found that, these closed film spots are conclusive for correct shape modeling, but making them enough sparse is important so that they are not similar to visible edge.

Measure: once we generate the distribution of hypothetical model, just corresponding analog image is compared with real MIcrosope image.MIcrosope image be carry out in advance to use basic curvature to produce the bianry image of cell membrane as basic method and Threshold Analysis afterwards.Use the accuracy of symmetrical brachymemma chamfering distance estimations comparison, after it, be used to as each particle specified wt or possibility.

Upgrade: after specified wt, select pro rata particle to produce the particle new for a group of next one repetition with their weight.This pays close attention to the distribution of particles in high likelihood region.Abandon having the particle of low possibility, and increase the particle with high likelihood.Use low variance method to carry out particle resampling.

As discussed in text, once embryo is modeled, we just can extract dynamic imaging parameter, for example, time between duration and the mitosis of cytokinesis.Our cell tracker software is applied to Matlab before and evaluation time (depends on the number of particle) in two seconds at every image to the scope of half a minute.The number that our existing software version is applied to C and depends on particle computing time is in the scope of 1 to 5 second.

embodiment 1

Determine the graphical analysis of embryo's developmental potentiality.

method

By 1 freezing cell human embryos, also referred to as zygote, thaw and put into nutrient culture media and those conditions of for example using in IVF program at condition of culture under cultivate.As above described in further detail, when they were frozen and therefore when indistinguishably freezing preservations in the 2PN phase, these embryos show it is the Typical Representative of (IVF) in vitro fertilization group.This embryo compared with freezing preservation in late period who grows after shifting with those embryos conventionally during being found in the fresh cycle with E.B.B. is contrary.For some experiments, embryo is placed in standard culture.For other experiments, embryo cultivates in the customization double dish with optical quality micropore.

Afterwards by growth embryo, 1 to 30, common every ware, the assembling of computerizeing control for digital image storage and analysis microscope time difference imaging separately.In some instances, use the inverted microscope of assembling warm table and incubator to carry out time difference imaging.In other examples, the mini microscope array that use is suitable for the customization of conventional couveuse inside carries out time difference imaging, and it can cultivate and the restriction of variable size minimum interval between consecutive image not being caught to adapt to hyperchannel many wares sample in same couveuse simultaneously.Use multiple microscopes to eliminate equally the needs to mobile example, it has increased the reliability of system accuracy and whole system.Imaging system is used dark field illumination, and it provides the image comparison of enhancing for feature extraction and graphical analysis subsequently, although have been noted that other illuminations have been enough.By separated from one another the single microscope in couveuse, its oneself controlled environment is provided to each double dish.This allows ware to proceed to and produce and do not destroy the environment of other samples from image space.

Collect time difference map picture and be used for cellular morphology Epidemiological Analysis subsequently, comprise at least one the measurement in following cell parameters: the duration of cytokinesis for the first time, for the first time and for the second time time interval between cell division and for the second time and for the third time time interval between cell division.The image showing in figure is nearly within 5 or 6 days every 5 minutes, to obtain with the time shutter of 1 second.As below more described in detail, cytokinesis is for the first time occurred one day after and is continued approximately 14 minutes by the fertilization of being everlasting.For the first time and the common interval of cell division for the second time on average approximately 11 hours.For the second time and the common interval of cell division for the third time on average approximately 1 hour.Therefore, imaging is a period of time that continues approximately 36 hours (adding deduct several hours) at after fertilization.

result

In the time period of six days, confirm the development time line (Fig. 2) of healthy human PIE in cultivation by time difference imaging.Observing normal human subject zygote divides at the 2nd day early experience First cleavage.Subsequently, before within the 4th day, combining closely as mulberry fruit tire, embryo was split into respectively 4 cells and 8 cell stages at the 2nd day and the 3rd day.When all can blastomere being divided into while producing the inner cell mass that to grow for embryo and ectogenesis in the trophocyte of the embryonic structure that is similar to placenta or body be multipotential stem cell, during forming, blastocyst within the 5th day and the 6th day, observes obvious Cell Differentiation on initial morphology.

Next we are four independently distributions of experiment normal and embryo of stagnating during the sample that the embryo of 242 normal fertilization of middle tracking and confirmation be cultured to the 5th day or the 6th day is set.In 242 embryos, by 100 Embryo Culture to the 5 days or the 6th day and observe blastocyst and form speed between 33%-53%, form speed similar (Fig. 3) to the blastocyst in typical IVF clinic.Remaining embryo stagnates at the different times of growing, and is everlasting between 2 cells and 8 cell stages most and is defined as undesired (Fig. 3).In order to determine that prediction embryo is successfully developed to the quantitative imaging parameter in blastocyst stage, we extract and have analyzed the several parameters from time difference video recording, comprise time interval between blastomere size, oolemma thickness, broken degree, the length of first cell cycle, several leading mitosis and mitotic duration for the first time.During growing normal and abnormal embryo's video image analysis, the embryo that we observe in many stagnations experiences abnormal cytokinesis during cell division for the first time.Normal embryo is completing cytokinesis from the narrow time window of 14.3+/-6.0 that separate completely that occur to daughter cell of cleavage groove minute, with steadily but controlled mode.This is shown in Fig. 4 top.On the contrary, undesired embryo shows in two abnormal cytokinesis phenotypes conventionally.In gentle phenotype, the morphology of cytokinesis and mechanism are seemingly normal, but the needed time of complete process is longer, from extra a few minutes to one hour (Fig. 4).The embryo of the cytokinesis that once in a while, experience extends a little still grows for blastocyst.In more several phenotypes, the morphology of cytokinesis and machine-processed multilated.For example, as shown in the embodiment in lower group of Fig. 4, embryo forms the cleavage groove of one side and experienced a series of abnormal film edge surge events several hours before finished breaking is less part.Observe equally other variations of these behaviors.In addition, prove that abnormal embryo of these more serious phenotypes usually becomes fragment, it may be the accessory substance that causes subsequently the abnormal cytokinesis of abnormal embryonic development that embryo's fracture is provided.

The labor of the image result to us showed before embryonic gene activation (EGA) starts, in the cytokinesis of interkinesis and mitosis, fetal tissues is followed the strict time in early days, and the developmental potentiality that shows embryo is to be predetermined by hereditary maternal program.Especially, we notice three temporal intervals or the parameter that in cell cycle of embryo in early days, are are strictly regulated and controled: (1) cytokinesis for the first time duration, (2) are for the second time and for the third time mitotic synchronism of the time interval between mitosis and (3) for the first time and for the second time.Relation between this three time intervals and morphological change is shown in Fig. 5, for fetal tissues, we measure these parameters and are respectively about 14.3+/-6.0 minute, 11.1+/-2.1 hour and 1.0+/-1.6 hour (being given as average positive/negative standard deviation herein).

We are that imaging is carried out in the embryo's of the 3PN (triploid) that starts from one cell stage small set (n=10) to fresh (not freezing preservation) equally.3PN embryo has shown that the timeline of following the boundary mark event identical with fresh normal embryo is until three cell cycles at least.Before our main experiment by these embryo's imagings in case checking imaging system (but due to technical reason not to blastocyst carry out).In this fresh embryo's set, 3 embryos follow the event time line similar to our 2PN embryo of freezing preservation, there is the duration of the cytokinesis of from 15 to 30 minutes scopes, for the first time and for the second time time between mitosis within the scope of 9.6 to 13.8 hours, for the second time and for the third time time between mitosis within the scope of 0.3 to 1.0 hour.But in 7 embryos, we observe unique cytokinesis phenotype, it is characterized by that 3 cleavage grooves occur simultaneously, cytokinesis extends a little and be finally split into 3 daughter cells (Fig. 4).These embryos have the time for the second time and between mitosis for the third time (4 cell to 5 cell) within the scope of the cytokinesis duration (being characterized as the initial of cleavage groove until be split into 3 times between daughter cell completely) within the scope of from 15 to 70 minutes, the time and 0.3 to 2.6 hour for the first time and between mitosis for the second time (3 cell to 4 cell) within the scope of 8.7 to 12.7 hours.Together with the different range of this observation and the cytokinesis phenotype being showed by undesired embryo, show not to be frozen in our embryonic development of freezing preservation preserve process lag and behavior similar to the fresh zygote that is split into two blastomeres.

Can predict by thering is cytokinesis for the first time between approximately 0 to 33 minutes, for the first time and for the second time for the second time and for the third time time between mitosis between the time between mitosis and 0 to 5.8 hour between 7.8 to 14.3 hours the embryo who reaches blastocyst stage, there is respectively 94% and 93% susceptibility and specificity (Fig. 6).On the contrary, show the predicted stagnation of embryo of the value outside one or more in these windows.All successfully fetal tissues that growth is blastocyst show similar value in all three parameters.On the contrary, abnormal embryo shows the variability (Fig. 6) of a large amount on they complete the length of the time that described interval spends.We observe (1) and indicate bad developmental potentiality than the normal longer time period that completes cytokinesis for the first time; (2) show to indicate bad developmental potentiality than normal longer or the shorter for the first time and for the second time interval between cell division; (3) show to indicate bad developmental potentiality than normal longer or the shorter for the second time and for the third time interval between cell division.Therefore, these parameters are that embryo is marched to the ability of blastocyst formation and the prediction of blastocyst quality.

Finally, although we notice that each parameter predicts embryo's developmental potentiality independently, the use of all three parameters provides the susceptibility and the specificity that all exceed 90%, has 3 times to the cut off of standard deviation.The recipient of these parameters operates sign (ROC) curve and is shown in Fig. 7.Curve in this figure shows the true positive rate (susceptibility) and false positive rate (1-specificity) contrast of various criterion deviation cut-off.In order to obtain this ROC, use time column number: true positive number=34 (correct Prediction reaches blastocyst); True negative number=54 (correct Prediction stagnation); False-positive number=4 (incorrect prediction reaches blastocyst); False-negative number=2 (incorrect prediction stagnation).

discuss

Our analysis is presented at the embryo who follows strict time in a mitosis of three spilting of an egg interkinesises and cytokinesis has much higher may growth to blastocyst stage and formation to have the high-quality blastocyst of the inner cell mass (ICM) of expansion.Dynamic form mathematic(al) parameter can be used to select the best embryo for the transfer in IVF program or freezing preservation.These parameters also can be used to differentiate the different qualities of blastocyst, allow the relative developmental potentiality classification of embryo in group.Standard practices in ICV clinic is to shift at 8 cell stages (the 3rd day).Some clinics are selected Embryo Culture to blastocyst stage (the 5th day), because blastocyst transfer doubles implantation rate with the highest compared with transfer in the 3rd day.But Extending culture is avoided in a lot of clinics, owing to the risk of the epigenetic disease increasing.Prediction imaging parameters can be used to predict embryo's viablity at 4 cell stages (the 2nd day) and before embryonic gene activation.This can allow than before morning whole one day of common implementation and marked change in their point subroutine of embryo experience by embryo's transfer or freezing preservation.This also can allow to select best embryo, for the analysis of PGD or other types.

embodiment 2

Verify that by gene expression analysis imaging parameters and gene expression analysis determine the purposes of developmental potentiality.

method

By 1 freezing cell human embryos, also referred to as zygote, thaw and put into nutrient culture media and those conditions of for example using in IVF program at condition of culture under cultivate.For some experiments, embryo is placed in standard culture.For other experiments, embryo cultivates in the customization double dish with optical quality micropore.

Embryo is shifted out and is collected as single embryo or the individual cells (blastomere) for gene expression analysis from nutrient culture media and imaging system.Each plate comprises embryo's potpourri conventionally, and some have reached the desired stage of development in results and other the stage of development early stagnates or be extensively broken.Those that have reached desired stage of development in results are classified as " normally ", and those of stagnation are considered to " abnormal ".For example, when the embryo of a plate being shifted out from image space second day late period in order to collect sample, reached 4 cell stages and any embryo of exceeding will be defined as normally, and could not reach 4 cell stages those will be marked as stagnation.The embryo of these stagnations is started to the puberty classification of stagnating according to them, will analyzed as being 2 cell stages of stagnation so that only there is the embryo of 2 blastocysts late period at the 2nd day.Carefully in sample collection get rid of on morphology seemingly dead and porose embryo's (blastomere of for example degenerating).Only show that the embryo's (normally with two kinds that stagnate) who lives is used to gene expression analysis.But they are allowed to grow to compared with may finally stagnating late period if show normal embryo when likely collection.Represent each embryo's the gene expression analysis in these kinds by quantitative RT-PCR (qRT-PCR).With the interval of about 24 hours, collect embryo for high-throughout qRT-PCR gene expression analysis from single imaging system, there is the multichannel reaction of nearly 96 genes to 96 sample determinations.Use Fluidigm Biomark system to carry out gene expression analysis, it can be received amount of rising and realize nearly 9216 and be simultaneously determined as basic qRT-PCR reaction with TaqMan.

result

In order to explain the basic molecular mechanism that may become morphology event, we are to relevant gene expression spectrum analysis.96 heterogeneic expressions that each sample evaluating belongs to a different category, comprise house-keeping gene, reproduction cell mark, maternal factor, EGA mark, trophoblast marker, inner cell mass mark, pluripotency mark, epigenetics regulatory factor, transcription factor, hormone receptor and other (table 1, in Figure 19).But in two different experiments are set, assess the gene of two different overlapping set, the unique gene sets of diagnosing human embryo destiny is provided.Described unique gene sets is from the data of the embryo about from model organism or hESC's gene expression and from we self unpub microarray data compilation.In this research, show for the first time the expression status of these gene sets in mankind's PIE.

Use geNorm method people (2009) Hum Reprod such as () ElToukhy T and AACt method people (2009) Nat Med 15:577-83 such as () Vanneste E to calculate each gene with respect to reference gene GAPDH and RPLPO and with respect to the expression value of gene mean value.Gene stability value and the coefficient of variation are 1.18 and 46% (for GAPDH) and 1.18 and 34% (RPLPO), are the most stable and just in time in the scope of typical allos sample set in 10 house-keeping genes of our test.In single blastomere, as expected, the each division of the amount of GAPDH and RPLPO transcript between 1 cell and 8 cell stages reduces about 1Ct value, owing to lacking EGA in the first three days that reducing by half of cleavage division affects and the mankind grow.In single blastomere, the expression of these reference genes is stable to maintenance between morula stage at 8 cells.In complete embryo's level, the Ct value of RPLPO and GAPDH in whole growth until morula stage keeps constant to a great extent.Expression at blastocyst stage RPLPO and GAPDH significantly raises, most possibly owing to there being the blastomere that increases number.These change does not affect RPLPO and the GAPDH reliability as reference gene.The puberty of great majority in the gene expression analysis carrying out in this research before focusing on morula stage, now the expression of reference gene is highly stable.

Differential gene expression between normal and undesired embryo.Fig. 8 demonstration is marked and drawed in radar map with logarithmic scale from the average expression of 52 genes of 6 abnormal 1 to 2 cell stages and 5 normal 1 to 2 cell stages.Conventionally the embryo who stagnates shows the amount of the minimizing of mRNA compared with fetal tissues, promotes cytokinesis, RNA processing and the biogenous gene of miRNA to be subject to the most serious impact.The gene representation highlighting with asterisk is as the determined significant difference statistically (p < 0.05) between normal and undesired embryo of Manny Whitney test.These 18 kinds of genes are Cofillin, DIAPH1, ECT2, MYLC2, DGCR8, Dicer, TARBP2, CPEB1, Symplekin, YBX2, ZAR1, CTNNB1, DNMT3B, TERT, YY1, IFGR2, BTF3 and NELF.Every kind of gene belongs to group as indicated in FIG., i.e. cytokinesis: Cofillin, DIAPH1, ECT2 and MYCL2; MiRNA is biological to be occurred: DGCR8, Dicer and TARBP2; RNA processing: YBX2; Maternal factor: ZAR1; House keeper: CTNNB1; Pluripotency: DNMT3B, TERT and YY1; Acceptor: IGFR2 and transcription factor: BTF3 and NELF.In most of examples, the expression of these genes ratio in normal 1 and 2 cell stages is higher in 1 and 2 cell stages of stagnating.

What is interesting is, some gene classification ratio in undesired embryo is more influenced in other.For example, in abnormal embryo, most house-keeping gene, hormone receptor and maternal factor change not at all in gene expression, but the related many genes of the biological generation of cytokinesis and miRNA demonstrate significantly reduced expression.And in affected gene, some gene shows than other much bigger difference between normal and undesired embryo.For example, the gene relating in the biological generation of miRNA path such as DGCR8, Dicer and TARBP2, shows the expression highly reducing in abnormal embryo.It should be noted that, CPEB1 and Symplekin, in the gene being had a strong impact on most two, belong to the identical molecular mechanism (Bettegowda, the people such as A. (2007) Front.Biosci.12:3713-3726) that regulates source of parents mRNA to store and pass through the activation of processing transcript poly (A) tail.These data show that the defect in embryo's abnormality and embryo's mRNA adjusting program is relevant.

Cytokinesis is associated with gene expression profile.With coding crucial cytokinesis component gene carry out gene expression analysis.By the identity of camera being installed on stereo microscope and the sample transfer process during nutrient culture media replacing and sample collection being made a video recording to follow the trail of each embryo.In the time of the undesired embryo's of assessment gene expression profile, we observe strong associated in the lower gene expression dose of abnormal cytokinesis and crucial cytokinesis composition.What is interesting is, undesired embryo's gene expression profile is the same with their dysmorphology phenotype is different and variable.

Found cytokinesis gene (Fig. 9) between normal 2 cell stages and undesired 2 cell stages and between normal 4 cell stages and undesired 4 cell stages (Figure 10) be different.Fig. 9 and 10 shows the relative expression of the gene of high expressed more in normal 2 cell human embryos (Fig. 9) and normal 4 cell stages (Figure 10) from different cytokinesis phenotypic correlation.As showed, during cytokinesis for the first time, show that 2 cell stages of the stagnation of abnormal film edge fluctuation have significantly reduced expression to all tested cytokinesis regulatory gene in Fig. 9.The gene that shows difference in Fig. 9 is anillin, cofillin, DIAPH 1, DIAPH2, DNM2, ECT2, MKLP2, MYCL2 and RhoA.Normal expression level provides with the post on the right and can find out in each gene higher.In photo above the chart of Fig. 9, show abnormal two cell stages, engineer's scale represents 50 μ m.Figure 10 shows the result of 4 cell stages of the stagnation of the abnormal cytokinesis from experience the cytokinesis that has one side cytokinesis ditch and greatly extend during first division, the expression of the reduction of described demonstration cytokinesis regulatory factor Anillin and ECT2.Engineer's scale in Figure 10 also represents 50 μ m.

Embryonic period, embryonic phase neural specific gene expression pattern.Figure 11 is presented at four special patterns embryonic period, embryonic phase (ESSP) of identifying during the gene expression analysis of single embryo to 141 normal developments and single blastomere.Be divided into the gene of each in four ESSP and be listed in (Figure 20) in table 2.Figure in Figure 11 by the expression pattern taking similar as basis by gene Clustering and on average producing their expression value (with respect to reference gene).Relative expression's level of ESSP is by average calculating the expression of the gene with similar expression pattern.Gene expression dose is done to figure to different cell stages, that is, and cell of 1c=; M=mulberry body, B=blastocyst.In Figure 11, the relative expression of the gene in each of four ESSP is shown as the function of growth, from 1 cell (1c) to mulberry body and blastocyst.ESSP1 shows matrilinear inheritance, and ESSP2 shows genetic transcription activation, and ESSP3 shows late activation and ESSP4 shows lasting transcript.As indicated in ESSP2, in IVF clinic, typical branchpoint occurs in the 3rd day, and now embryo is experiencing owing to the significant growth of embryonic gene activation and changing.Time difference imaging data instruction embryo's developmental potentiality can be identified by 4 cell stages, allows thus embryo the 2nd day and the transfer of morning before this gene activation.This early stage transfer is useful for the success ratio that improves IVF program.

Table 2 (Figure 20) is listed each the gene in four ESSP that belong to identified.To reference gene (GAPDH and RPLPO) and with respect to the relative gene expression dose of every kind of gene of gene mean value estimation.Contrast embryo's the expression pattern of every kind of gene of development time line is followed in following four ESSP: ESSP pattern (1) is early stage, start high, the gene that slowly reduces and closed before blastocyst; ESSP pattern (2) mid-term: the gene of opening after 4 cell stages; ESSP pattern (3) late period: the gene of opening in the time of mulberry body or blastocyst is constant with ESSP pattern (4): the gene with relative constant expression value.

ESSP1 describes the pattern of the gene of matrilinear inheritance.These transcripts start and subsequently along with embryonic development reduces as blastocyst taking the high expression level of zygophase.The half life period of these transcripts is about 21 hours.From for example GDF9 of typical maternal factor of other model organisms and ZAR1 and reproduction cell (egg mother cell) specific gene VASA and DAZL in this classification.ESSP2 comprises embryo's activating gene, and it transcribes first after 4 cell stages in embryo.Some gene indicator gauges in this classification reveal two ripples activation, for the first time and compared with little once at 5 to 6 cell stages and for the second time and larger once at 8 cell stages.From other the known EGA gene of model organism, for example EIF1AX31 and JARID1B32, in this classification.ESSP3 by until blastocyst stage the late activation just expressed genomic constitution, comprise trophoblast marker GCM1.ESSP4 is included in the lasting transcript that keeps stably express in whole growth with respect to reference gene.The half life period of these genes is 193 hours, is about 9 times than ESSP, and this classification comprises house-keeping gene, transcription factor, epigenetic regulatory factor, hormone receptor and other potpourri.In another experiment setting of 61 samples that uses single fetal tissues and blastomere, confirm these 4 kinds of patterns of gene expression.

The abnormal embryo who shows abnormal cyto-dynamics and mitosis behavior during first division is relevant with high unsettled gene expression profile, especially relates in the gene of embryo RNA management.Therefore, a kind of method can be in conjunction with these methodologies to provide the method that can be used to predict PIE viablity.Result shows that abnormal embryo starts life with the Defective program in RNA processing and the biological generation of miRNA, causes the excessive degradation of maternal mRNA.The random nature of these unadjusted RNA degradeds causes the random disruptions of transcript, causes the various abnormal phenotype of observing in undesired embryo.The miRNA of decline level causes the defect in the maternal RNA degraded of adjusting, causes the arrest of development at different times.

Single blastomere analysis.In order to assess in mankind's PIE when start molecule differentiation, analyze CDX2 expression from the single blastomere in budding 17 embryos results of difference.Figure 12 A shows that relative expression's level of two gene C TBBN1 (black post) and CDX2 (shallow post) is budding function, from 2 cells to blastocyst.As can be seen, in the single blastomere of the embryo from before 4 cell stages, CDX2 is accidentally expressed (Figure 12 A) with reduced levels.But since 6 cell stages, each embryo comprises at least one blastomere of expressing CDX2 with the level of signifiance.The expression that is also shown in the house-keeping gene CTNNB1 in Figure 12 A keeps constant in the blastomere from identical embryo, and the Achieve Variety pattern of instruction CDX2 is not the artefact of qPCR.From the similar observation of the data acknowledgement of independent experiment.These results show that the molecule differentiation of mankind's PIE can occur immediately as early as possible after 4 cell stages.

What is interesting is, the inspection of the gene expression profile in single blastomere is shown and comprises the embryo who has corresponding to the blastomere of the budding gene expression label of difference.Any given embryo's of any given time gene expression profile equal maternal mRNA degraded and EGA with.The younger blastomere of early development phase contains the maternal transcript of a large amount and the zygotic gene of low amount and the contrary budding more old blastomere in more high is suitable for conventionally.In this experiment, specific procedure is defined as the average expression value embryo program of 10 ESSP1 marks (maternal transcript) by the average expression of 10 ESSP2 marks (embryo's transcript).The maternal transcript group using comprises DAZL, GDF3, IFITM1, STELLAR, SYCP3, VASA, GDF9, PDCD5, ZAR1 and ZP1, and embryonic gene group comprises ATF7IP, CCNA1, EIF1AX, EIF4A3, H2AFZ, HSP70.1, JARID1B, LSM3, PABPC1 and SERTAD1.In 6 blastomeres that success is collected specific 8 cell stages from this, 3 blastomeres demonstrate to from the similar gene expression label of normal 3 cell stage samples, and all the other 3 blastomeres similar to the blastomere from normal 8 cell stages (Figure 12 B).The most probable explanation of this observation is the stagnation of the cell of a subgroup in embryo.In another 9 cell stage in the sample that this part stagnation phenotype is tested at us equally and 2 mulberry bodys, observe.Maternal transcript level is in the high fact still in the blastomere of the stagnation of the cost time quantum identical with their normal sister cell in cultivation, and the degraded that shows maternal RNA is not the spontaneous process of simple generation in time and the effect of most probable needs for example microRNAs of special RNA degradation mechanism (miRNA).These data provide maternal mRNA degraded to be growth event conservative between the mammal embryo emergence period and to be the needed further evidence of normal embryonic development (Bettegowda, the people such as A. (2008) Reprod.Fertil.Dev.20:45-53) equally.In addition, these data show that blastomere single in embryo is spontaneous and can grows independently of one another.In addition, the instruction of these results can be used gene expression dose test described herein with needing to be tested in tested cell the level of mRNA (its indicator expression), wherein RNA be known as maternal program a part gene and in the late period of embryonic development this developmental level continue relevant with the possibility of undesired result, or a part for embryo's program, wherein shortage is in time indicated the possibility of undesired result.The maternal program gene of inspection is ZAR1, PDCD5, NLRP5, H5F1, GDF9 and BNC2 herein.Other the maternal gene that affects is known and available.

Embryonic gene activation.This method is the basis that is found to be that the embryo with abnormal arrest of development conventionally shows abnormal cytokinesis and mitotic time during a previous tertiary split occurs EGA (embryonic gene activation) at least partly.This destiny that shows embryonic development is mainly determined by matrilinear inheritance, a significantly consistent discovery of the mathematical model of growing before implanting with the mankind that carried out in 200134 by people such as Hardy.And cytokinesis and mitotic abnormal with regulate the biological generation of miRNA and maternal mRNA covers, the level of the reduction of maternal transcript is strongly relevant in the gene of storage and reactivation.MiRNA, by promoting the mRNA in different bioprocess to degrade to regulate translation, comprises biological development and differentiation (Blakaj, A. & Lin, H. (2008) J.Biol.Chem.283:9505-9508; Stefani, G. & Slack, F.J. (2008) Nat.Rev.Mol.Cell Biol.9:219-230).Show that from the evidence of the increase of model organism miRNA can be the crucial regulatory factor (Bettegowda, the people such as A. (2008) Reprod.Fertil.Dev.20:45-53) of maternal transcript degraded in embryo in early days.Therefore, the defect that miRNA is biological to be occurred will probably cause abnormal embryonic development, on the other hand, can not cause equally bad embryo to occur by the maternal mRNA of correct management.Mammal ovocyte synthesizes supports the needed a large amount of maternal rna transcription things of body early embryo growth before mother produces.These transcripts suppressed and storage one elongated segment time, until they fertilization after be re-activated.Defect in this maternal RNA supervisory routine may affect the amount of maternal transcript and quality and therefore damage the chance of successfully growing.

The model of assessment embryo viablity.The imaging that Figure 13 shows to be correlated with and analysis of molecules are the model that basic human embryos are grown.Shown is comprise prediction successfully develop into blastocyst crucial blink be developed to the timeline of blastocyst and the diagram of embryonic development from zygote.As illustrated, key molecule data instruction human embryos start life with heredity from the egg mother cell RNA of mother's different sets.The RNA of this set keeps and packaging by special RNA supervisory routine in ovum.After fertilization, the degraded of maternal RNA to special one sub-set of ovum (ESSP1: embryonic period, embryonic phase special pattern 1) must be accompanied egg mother cell and be reduced to the beginning of embryo's transformation.Meanwhile, other RNA distributes to each blastomere ideally fifty-fifty with growing continuation (ESSP4).The successful degraded of RNA and distribute transcribing and finish with the embryonic gene group activation (EGA) of the spontaneous form of cell and the gene of ESSP2.It should be noted that during cleavage division, embryo's blastomere can be stagnated or carry out independently.In embryo, the result of the spontaneous growth of cell is that single blastomere can be stagnated or carry out and in the time that 8 cells proceed to morula stage and exceed, the impact of the number of the cell of 8 cells be stagnated or be exceeded to blastocyst quality will.Imaging data confirms to have to be predicted successfully or key period of failed growth: cytokinesis for the first time, cleavage division for the second time and for the second time and the synchronism of cleavage division for the third time.Cell tracker algorithm and the software before these parameters can be used, described are measured automatically.The transfer (before EGA) that described system and method is used to use crucial imaging predictor diagnosis embryo outcome and can allows less embryo more early stage in growth.

embodiment 3

By oocyte maturation and embryonic development imaging subsequently.

result

One in the major limitation of existing IVF operation is Oocyte quality and availability.For example, from minor cycle pond, (small cyclic pool) supplements egg mother cell to existing IVF scheme, is provided for a small amount of egg mother cell (for example 1-20) of fertilization.And it is jejune and conventionally because the potentiality of the embryonic development reducing under existing condition of culture and being excluded that approximately 20% the egg mother cell obtaining after IVF operating period hormonal stimulation is classified as.

A kind of method that increases egg mother cell pond is to pass through maturation in vitro.Figure 14 shows three periods of growing during maturation in vitro, comprise germ-vesicle, mid-term I and mid-term II.Germ-vesicle and I phase in mid-term be defined as immature egg mother cell and mid-term II because the existence of the ripe first polar body occurring for 24-28 hour after initial and be defined as ripe in vitro.Figure 15 shows by the embryonic development of the egg mother cell of maturation in vitro.

Another method that increases egg mother cell pond is to supplement egg mother cell from primary and secondary pond, and the egg mother cell that reaches several thousand is provided.In this operation, dormancy folliculus supplements and carries out vitro procedure from ovary has the combination of normal dyeing body, epigenetic state, rna expression and morphologic egg mother cell to produce.On the other hand, egg mother cell can derive from pluripotent stem cell that vitro differentiation is reproduction cell and ripe be human oocytes.

As illustrated in fig. 14, the maturation in vitro process of egg mother cell is carried out mark for some cellular change of the cell parameters measuring and analyze in the method for the invention by being used to definition.These comprise, for example, the morphologic variation of oocyte membrane, such as the speed and the degree that separate from oolemma; The morphologic variation of oocyte nuclei, such as the startup of germinal vesicle breakdown (GVBD), complete and speed; The motion of the speed of the motion of particle and direction and first polar body and discharge in tenuigenin and nucleus.

embodiment 4

By Stem cell differentiation imaging.

result

Time difference imaging analysis also can be used to viablity, potentiality of development and the result of the cell of assessing other types, such as pluripotent stem cell (iPSC) and the hESC (hESC) of stem cell, induction.The developmental potentiality of stem cell can be by assessing to measure cell development and morphologic variation (Figure 17) between the idiophase with time difference graphical analysis.Can analyze and select afterwards the cell for the differentiation of transplanting or other purposes in body.Can extract and analyze from time difference imaging data several parameters of stem cell, motion, schizotype, differentiation, asymmetric division (now a daughter cell keeps stem cell and another daughter cell differentiation), symmetrical division (now two daughter cells all remain stem cell or two all differentiation) and the destiny specialization (determining before in the time of Stem cell differentiation) of for example, time, cell size and shape between duration, the mitosis event of cytokinesis, the number of cell, cell.

The basic formula of stem-cell therapy is that undifferentiated stem cell can in vitro culture, is divided into specific cell type and migrates to acceptor subsequently so that injured tissue and/or neomorph.Time difference imaging analysis can be used as high flux non-intruding device to identify the offspring's who forms the differentiation that can be incorporated into the non-carcinogenic in mature tissue stem cell.Possible application comprises the treatment of for example Alzheimer disease of sacred disease and parkinsonism, vascular system disease and heart disease, muscle and skeletal diseases such as arthritis, autoimmune disease and cancer and passes through drug development and the novel therapeutic of evaluation index.

In the mankind, impaired tissue replaces by the supplementary and differentiation continuing of stem cell in body conventionally.But the ability of health regeneration reduced along with the age.Such a example is by the damaged urinary incontinence causing of sphincter.Age it is believed that it is in the main reason that sphincter is damaged, because the number of meat fiber and neural density reduce in time.In order to treat the patient who suffers from incontinence, iPSC can derive from the fibroblast of cultivating from vaginal wall tissue to produce the smooth muscle cell of differentiation.The cell of these differentiation afterwards can be transplanted in body.Before transplanting, time difference imaging analysis can be used to iPSC with regard to versatility, break up, methylate and carcinogenicity characterizes.Other application comprises the time difference imaging (Figure 18) that derives from the patient's who suffers from parkinsonism Skin Cell and be divided into the neuronic iPSC for transplanting.

embodiment 5

Confirm imaging parameters by automatic analysis

As our time difference view data is proved, it is that process and the embryo of height change between embryo of the same age can show various behaviors during cell division that human embryos are grown.Therefore, for example manual sign of the duration of highly abnormal cytokinesis (Fig. 4) of some growth event can be explained.In order to confirm our imaging parameters and systematically to predict the ability that blastocyst forms, we have developed for automatic tracing cell division until the algorithm of 4 cell stages.Our tracing algorithm uses taking sequential Monte Carlo method as basic probability model assessment technology.This technology is supposed the distribution of embryo model, is compared to move taking simple optical model as basic analog image and by these simulations that (Figure 21 a) with the view data of observing by generation.

Embryo is modeled as there is position, the oval set (to represent the relative height of cell) of direction and overlapping index.About these models, can extract the time between duration and the mitosis of cytokinesis.Cytokinesis defines by appearance first (depression forms along spilting of an egg axle when the two poles of the earth) to the separation that completes daughter cell of cytokinesis ditch conventionally.We are by problem reduction the duration by cytokinesis being roughly estimated as to the cell elongation before 1 cell to 2 cell division.If the gap on shaft length exceedes 15% (empirically selecting), cell is considered to extend.Time between mitosis is directly by extracting cell count in each model.

We have tested our algorithm on one group of 14 people embryo, and (Figure 21 b) and will measure automatically with manual image analysis and compare that (Figure 21 c, Figure 21 are d).In this data group, 8 in 14 embryos have reached and have had the good morphologic blastocyst stage (Figure 21 e, upper figure).Automatic measurement coincide very much with manual measurement and all 8 embryos are reached blastocyst by correct Prediction.Two in 14 embryos reach and have bad morphologic blastocyst (inner cell mass of bad quality, Figure 21 e, figure below).For these embryos, manually assessment show 1 will reach blastocyst and 1 will stagnate, and measurement is predicted and both will be stagnated automatically.Finally, 4 in 14 embryos stagnated and all correct Prediction stagnations of two kinds of methods before blastocyst stage.

Particle filter framework

Particle filter is taking Monte Carlo simulation as basic modeling technology.The distribution of the model that it is used to suppose by generation and these models and viewed data are relatively simulated to unknown or " hiding " model.It is to follow the trail of fissional ideal candidates person that its ability that adapts to arbitrary motion dynamics and measuring uncertainty makes it.

Particle filter is followed the trail of the increment of three primary variabless in time: state x, contrast u and measurement z.State variable x is that we wish the embryo's who assesses model and are represented as ellipse (2D) or the set of ellipsoid (3D).Collative variables u is the input that state variable is transformed and is made up of our cell proliferation and branching model.Measurand z is the observation of state and the image construction that obtained by time difference microscopy by us.These parameters are described in part below in further detail.

Represent the estimation of the existing state x of each time step t with posterior probability distribution.This posterior probability is commonly called the image measurement z that puts letter (the belief) and be defined as given all past _1:tcontrast u with the past _1:tstanding state x _tconditional probability

bel(x _t)＝p(x _t|u _1:t，z _1:t).

Particle filter is roughly estimated, rear, to be expressed as with sample or the particle of one group of weighting:

x_{t} = x_{t}^{[1]}, x_{t}^{[2]}, . . ., x_{t}^{[M]},

Wherein M is the number of particle.Term particle and embryo model are used interchangeably herein.Therefore, single particle xt ^[m]a hypothesis of embryo model when (wherein 1 <=m <=M) is time t.

After initial, three steps of particle filter repeated application.First step is hypothesis, and wherein each particle rises in value with contrast input:

The set of the particle obtaining is the approximate value of described prior probability.Second step is to measure to upgrade, the wherein weights of importance of the designated probability corresponding to existing measurement of each particle:

The set of the particle of described weighting is the approximate value of posterior bel (xt).

The principal ingredient of particle filter occurs in the 3rd step, and wherein the set of particle is according to their weight resampling.Distribution of particles in the region of this resampling step concern maximum probability.

Cell represents

Cell is expressed as to the ellipse in 2D space.Each cell has 2 in main shaft, minor axis and Cartesian coordinate dimension position, by under establish an equation and provide:

Each ellipse has direction θ (deflection) equally, and this allows it in x-y plane, to rotate.Because ellipse almost always overlaps each other, we indicate overlapping index h equally, and it specifies overlapping order (or relative height of cell).Therefore the parameter of each embryo model when time t is given as:

Wherein N is the number of the cell in this model.

Cell is upset and division

The first step of particle filter is prediction, wherein uses contrast input by each particle increment.For our application, there are us to want the behavior of two types of modeling.The behavior of the first type comprises cell movement, and it comprises translation, about the variation in the rotation of deflection angle and the length of main shaft and minor axis.The behavior of the second type is cell division, and one of them cell division is two new cells.

For by cell movement modeling, our contrast input adopts each value of particle and the each cell of random permutation: x _0i, y _0i, a _i, b _i, θ _i.Described upset is (is conventionally set as initialized value 5%) that uses that relatively little variable samples immediately from normal distribution.

For by cell division modeling, we have used following method.Given point in time, for each particle, we suppose 50% probability that in cell will divide.This value be select through experience and cross on a large scale possible cell division and keep the good covering to existing configuration simultaneously.Expect if divided, the cell of so described division is random selection.More complicated model will be considered for example number of cell and these schizotypes historical and may be taking the viewed behavior from True Data as basic production model in particle of other factor.

In the time of the selected division of cell, use the symmetry division along oval main shaft, produce two daughter cells of same size and shape.Each value of random permutation daughter cell afterwards.From normal distribution but with larger variable to upsetting again sub-sampling (initial value 10%) to adapt to variability large new cell shape.Finally, the overlapping index of two daughter cells is selected at random and is kept their common overlapping with respect to remaining cell.

Image simulation

After contrast input is applied to each particle, particle represents to be transformed in the image of the simulation that can compare with true picture.Accurate image simulation may be difficult task and usually need to use ray tracing technique and optical model.Method of the present invention focuses on the simulation feature of easily identifying in image instead of attempts to simulate real image.Especially, the image of cell membrane is simulated.

There are two entities must considering to observe.First, single-phase upper although microscope focuses on by embryo, the degree of depth of visual field is very large and from whole embryo collection light out of focus almost.Secondly, and, embryo is by partially transparent, it means the film of cell of embryo bottom, and (but not always) can be in sight by the cell at embryo top sometimes.

Consider these entities observations, now described is image simulation model.For each cell, use overlapping index h that corresponding elliptical shape is projected on the image of simulation.Corresponding pixel value is set to 1 bi-values and expands can be with observed to the film thickness compared with view data to produce.Overlapping index h designated cell is positioned at the order at mutual top.Because closed cell membrane is only sometimes visible, if detect closed point, they are placed on and have low probability in the image of simulation of (conventionally about 10%) so.In fact, when these closed film spots are while being necessary for accurate shape modeling, importantly make they enough sparse so that they can not be similar to visible edge.

Image preprocessing

Measurand z will be described now.The object of method of the present invention be from MIcrosope image extract cell membrane binary picture for simulation image ratio.These films show higher curvature and high contrast, extract but use the thresholding technology of density or color basis and be not easy.Therefore, used principal curvatures for basic detecting device.This method is used Hessian operator:

Wherein, Ixx, Ixy and Iyy, be the secondary partial derivative in pixel site s and Gauss's yardstick σ estimation.The eigenwert of 2x2 Gauss matrix provide about principal curvatures information, and the symbol of eigenwert makes a distinction " paddy " and " ridge " 43.In order to detect bright peak or ridge, the principal curvatures of each axle is calculated as

P(s)＝|min(λ ₂，0)|，

Wherein, λ 2 is minimal eigenvalues.In order to detect the film of different-thickness, having used the yardstick of covering certain limit (is σ _min<=σ <=σ _max) Hessian operator and extracted the maximum curvature of whole this scope.The bianry image of the cell membrane that finally, Hessian image is extracted with generation by thresholding.Threshold level is set to double the standard variance of the axle pixel value in Hessian conventionally.

Particle weight

As be entitled as described in the part of " particle filter framework ", second key step of particle filter is to measure to upgrade, and wherein specifies the weights of importance of particle corresponding to the probability of the existing measurement of given particle model.In our example, weights of importance be by by the MIcrosope image of preprocessing discussed above with same be that analog image discussed above is compared and determined.

Studied this problem, wherein particle filter weight is by comparing the image of simulation to calculate with real image by normal interactive information before.The idea that this mode is mated with occupancy grid is similar, and its searching is all occupied (value 1) or is all the pixel site of empty (value 0).But these methods have trouble at described imitation and actual image while not overlapping a little similar in shape.On the contrary, the method for describing is to be basic plausibility function in order to chamfering distance, and it measures the mean value from a point set to another minimum distance.Define two point set A (in true several destination aggregation (mda)s of size m) and B (in true several destination aggregation (mda)s of size n), respectively corresponding to the non-zero pixels in analog image and real image.Be given as from the chamfering distance forward of point set A to B:

Similarly definition chamfering distance backward.The inventive method is used symmetrical chamfering distance, and it provides and analog image is mated many good with true picture and true picture mates how well with analog image measures:

d _sym(A，B)＝d(A，B)+d(B，A).

In fact, single range observation is shortened to reduce the impact of noise.In order to reduce computing time, the pixel site during distance transforms by the distance of searching image is determined.

Chamfering distance is used as our possibility of DATA REASONING of given analogy model and measures.That is to say, at time t, for given image measurement z _twith particle model xt _[m], particle weights of importance is given as:

Constant λ is set to 1 and can change to control the planarity that possibility distributes conventionally.

Particle resampling and DYNAMIC DISTRIBUTION

In particle filter, the 3rd key step is resampling, the set of wherein particle and their weight being selected to produce new particle pro rata.The particle with low probability is excluded, and the particle with high probability is doubled.There are a large amount of previous works of exploitation for the efficient algorithm of resampling.This method is used low variable method.

A selection that major issue is number of particles in particle filter.The most simply selecting is to use fixed value, is called M=1000.For each time step, the set of M particle is transformed in another set of formed objects afterwards.In the application's context, cell be non-activity or only during the slight size changing and position, may have relative long a period of time.Utilize the advantage of this observation to process load by reducing according to the number of the amount dynamic assignment particle of cytoactive.That is to say, when cell be active and division time, we increase the number of particle and in the time that particle is non-activity, we reduce the number of particle.

In order to measure the degree of cytoactive, calculate new images (obtaining by microscope) and image before between picture element density the difference of two squares and (SSD).In order to reduce noise, first with Gaussian filter by image smoothing and use in time cause and effect moving average by level and smooth the value of SSD.Afterwards by the number of particle and this value dynamic adjustments and shortening to rest in boundary line 100 < M < 1000 pro rata.Figure 30 is how the number that shows particle can be allocated for from the chart of embryo's division of 1 cell to 4 cell stage.What it should be noted that is the measurement that this method only provides the amount of " activity " in image, but does not distinguish cell division and embryo movement (shifting and/or rotation), because the image registration before not carrying out.In this situation (determining the number of particle), this is acceptable, because the number of particle all should increase in any event.In fact, in most probable embryo model, we have adjusted the number of particle equally as basis taking the number of cell.That is to say, when more cell it is believed that while being present in image, produce more particle.

The limitation that two dimension is followed the trail of

Above-described 2D cell tracker algorithm is used to determine the number of cell in embryo and their 2D shape.But its fact that is not had potential entity to represent is limited to.This may be or may not be important to measure embryo viablity for automatic tracing cell division.For example, the time between duration and the cell division of for example cytokinesis of some parameter can measure with 2D cell tracker algorithm.In next part, we are 3D by our 2D model extension.In order to process the closure and the degree of depth uncertainty that produce from 2D image simulation 3D shape, geometrical constraint and constraint condition to cell volume concentration are used.

Cell represents and three-dimensional tracking

This part has been described the algorithm of following the trail of for fissional 3D.From many extending in this algorithm in the step of 2D algorithm, there is the exception of several keys.The new cell that is useful on 3D purposes represents.Cell is represented as the ellipsoid in 3d space now, by following formula:

Each ellipsoid has direction θ, pitch ψ and rolling α equally.Therefore, the expression of each cell model of time t is given as:

A material impact of the model of this revision is possible have the uncertainty relevant with the 3D shape of inferring from 2D image.For example, be in shape the cell of spheroid will have the outward appearance similar with the cell of longer pitch rotation to thering is longer main shaft.This is not main important thing, because as by as shown in below, distribution of particles will keep these multiple hypothesis until for example can obtain enough information, to produce difference (from event such as cell division).

It is tight that ellipsoid is considered to; That is to say, distortion can not be by clearly modeling.But we allow a small amount of overlapping between adjacent ellipses body and in these overlapping districts, we suppose that cell is to being complanation each other.This is major reason, because it is conventionally observed in embryo and we explain it in following part.

Cell is upset and division

Our 3D cell division is similar to the model in part 4 " cell is upset and division " with upset model, has a few exceptions.The simulation of 3D shape can be used to force volume conservation.It is large arbitrarily that this prevents that Growth of Cells from obtaining, especially in z direction.Volume conservation is used to two kinds of situations.The first, for cell upset, single shaft a and b be change and calculate c so that the volume of individual cells is kept.The second, for cell division, use following constraint condition:

Wherein subscript p indicates mother cell and subscript d1 and two daughter cells of d2 instruction.In fact, we by make embryo's cumulative volume initial volume positive/negative 5% between fluctuation allow slightly to violate these constraint condition.This is used to compensate the possible inaccuracy in initial volume simulation.

In the time of the selected division of cell in 3D, its division is modeled in the following manner.First, for selected individual cells, use the division along oval-shaped major axis, it can be a, b or c, depends on configuration.Consider the rotation of mother cell, daughter cell is initialised to equate in the uniform parts in size and space.Reuse afterwards the normal distribution with 10% the variable that is set as initial value their parameter is upset to cover possible configuration on a large scale.

Geometrical constraint

Closed and the probabilistic problem of the degree of depth partly relaxes by the conservation of volume.But, equally need to be about the constraint condition of adjacent ellipsoidal spatial relationship.First constraint condition is that cell must not overlappingly on diameter exceed 20%.For the cell of overlapping acceptable amount, them are done to the hypothesis of complanation each other.Described particle model intersects ellipsoid inside point by ignoring during image simulation represents this phenomenon.This is that experience excites and well relevant to entity observed behavior.

Force the second constraint condition that keeps cell very approximate.This constraint condition is directly relevant with human embryonic entity behavior, and wherein cell is called as the film constraint of oolemma.Described band is modeled as spherical shell and uses it to force boundary condition.The radius of described band is set to than the radius of 1 cell stage large 30%.

As below implemented these constraint condition.As described above, for each particle of preset time, random contrast input is used to produce new particle.As any in sporocarp constraint condition is breached, new particle is excluded and uses new random contrast.If do not produce satisfied new particle after the trial of some, particle is excluded so.

Image simulation

In embodiment, the advantage of dark field illumination used is that cell membrane is than the more light of cell interior scattering.This impact is the most remarkable in the cell membrane site parallel with optical axis (z-axle).Therefore, for analog image, in our 3D model, search these sites because their rotation its be not must be positioned in ellipsoidal equator.Consider visible and closed edge, as discussed above, follow afterwards similar rule.

Cell tracker embodiment in 2D

This embodiment and automated cell microscope about and use the above-described algorithm of following the trail of for fissional 2D.Design this model with the number of cell in tracking image and the 2D profile of cell membrane.The first step is Image Acquisition, and it excites for example image simulation of part and image preprocessing subsequently.Obtain the time difference image sequence for this embodiment with the Olympus IX-50 inverted microscope of the customization with 10X object lens.Microscope is adjusted for dark field illumination, and wherein the light cone of hollow focuses on sample by place circular hole between light source and collector lens.Object lens are collected by the light of sample scattering and direct reflection and transmission light, produce bright image on dark background.The advantage of dark field illumination be cell membrane often than the more light of cell interior scattering, increase thus their contrast.Equip microscope with the incubator of warm table and customization and in the time period that reaches 5 or 6 days, cultivate embryo with permission.Catch image by the Olympus SLR digital camera images that is erected at IX-50 side with the interval of 5 minutes.

Being imaged on when they are zygote or the zygote with rough spherical form of embryo starts.For by the set initialization of particle, as the Hessian of the calculated threshold of describing in the 6th part " image preprocessing " and use least square fitting circle.Use afterwards the random orientation from being uniformly distributed sampling that all particles are initialized as to circle.

Figure 31 shows for following the trail of from the result of the fissional 2D algorithm of 1 cell to 4 cell stage.Result showed cell film successfully extracts by described algorithm, or even for the cell of the part closure towards bottom.What it should be noted that is in the application of most of particle filters, " single " best model be usually represented as from the weighting of the state parameter of distribution of particles and.But, for result herein, show the particle with maximum probability.

Cell tracker embodiment in 3D

Figure 32 shows above-described for following the trail of from two successfully application of the 3D algorithm of 1 cell to 4 cell stage.Figure 33 is the chart that shows the embodiment (first embodiment showing in corresponding to Figure 32) that how to distribute of particle during 1 cell to 2 cell division.This figure shows the 3D position of each cell centre.When cell starts division, which daughter cell predictive display goes out about will be positioned at the uncertainty at another top, but this is solved in a pair of framework.

Extract Prediction Parameters

Once the method for describing before using, just can be from some parameter of model extraction by embryo's modeling.Conventionally, use preferably or the model of maximum likelihood.These parameters for example comprise the duration of cytokinesis for the first time, for the first time and for the second time time between cell division and time between cell division for the second time and for the third time.Extended the duration of how long carrying out roughly to estimate cytokinesis before it is split into two cells by the model of measuring cell.Elongation can be measured by the ratio of paying close attention to oval-shaped main shaft and minor axis.Can comprise from other parameters of model extraction the angle, broken etc. of fertilization and the shape of the time between cell division, cell and symmetry and fission process, division for the first time.Can use 2D cell tracker algorithm or 3D cell tracker algorithm extracting parameter.

Occuring to first daughter cell by cytokinesis ditch completes to separate and defines cytokinesis.Because our embryo model is made up of indeformable ellipse, the appearance of qualification cytokinesis ditch is challenging task.One method will allow oval distortion, but this causes more complicated tracing problem based.Another kind method will be the variation of finding the MIcrosope image mean curvature of preprocessing; But this has destroyed the object of making great efforts directly to measure from embryo model our Prediction Parameters.Therefore, we by problem reduction the duration by being roughly estimated as to the cell elongation before 1 cell to 2 cell division the duration of cytokinesis for the first time.The ratio of extending by calculating oval-shaped main shaft and minor axis is come quantitatively.If:

\frac{a - b}{b} &GreaterEqual; 15 %,

Cell is considered to extend.

15% this value be experience select and for this specific data acquisition operational excellence; But can use other values.Once embryo model is split into 2 cells, we can extract by calculating duration of elongation of 1 cell model the duration of roughly estimating of cytokinesis for the first time.

The time of measuring between mitosis event in principle, is simple.For example, the time between mitosis is measured as the time between 2 cell models and 3 cell models for the first time and for the second time.But in some instances, embryo can show unusual and random behavior.This for example comprises and proceeds to 2 cells from 1 cell, the embryo who proceeds to obvious 3 cells or 4 cells and get back to afterwards 2 cells from 2 cells.Described algorithm can be followed the trail of the behavior of this type, but it has proposed challenge to determining the time interval between mitosis event.

A kind of mode of processing this behavior is as follows: replace time of measuring between 2 cells and 3 cell models (in order to find for the first time and time between mitosis for the second time), this can roughly estimate by the number of computed image framework (wherein 2 cell models are most probable) simply.This in some instances operational excellence but always do not represent the actual time between mitosis event.Can process these events by the dielectric imposed limits on basic model of the number taking cell.That is to say, when distribution from each iteration is selected preferably or when most probable model, can need the number of the cell in model always keep identical or increase, but never decline.Force after this constraint condition, the time of calculating between mitosis event is simple.This constraint condition is also filtered for the tracking result that may show a small amount of shake, and for example it can occur once in a while in the time that model switches back and forth between 1 cell and 2 cell models.

For following the trail of the method for Prediction Parameters

Figure 35 shows the process flow diagram of having summarized above-described method.How process flow diagram can analyze single embryo (although this can be used to cell and the stem cell of multiple embryos or other types) if showing.In the first step, obtain embryo's image (" measurement ") by time difference microscopy.This image can be saved to file and the time point below reopens.Image conventionally by preprocessing to strengthen some feature, although this not necessarily.Predict the model of possible embryo's configuration and from these modeling images (" prediction ").The image of simulating can comprise the image of cell membrane described above or represent more accurately the image of preprocessing MIcrosope image before.Afterwards model is compared with the MIcrosope image of preprocessing (" comparison ").Use this relatively, retain best prediction, get rid of bad prediction simultaneously.The set of the prediction that obtained is afterwards used to improve the prediction to next image.After multiple consecutive images are carried out to this process, be directly possible from best one or more model measurement Morphologic Parameters, for example, time between the duration of cytokinesis and mitosis event.As previously discussed, these parameters can be used to assess embryo's viablity.

Embodiment 7

The automatic analysis of cytoactive

Above-described method need to be followed the trail of cytocerastic ability via microscope.For embryo, desired is to follow the trail of the multiple embryos that cultivate together in same ware.Analytical approach used herein need equally the image that the cycle obtains (for example for the every 1-30 of embryo minute once, continue 1-5 days; The different time intervals can be used to the such as stem cell of cell of other types).Therefore design formation method with automatic tracing embryonic development.

In time difference microscopy, cell is grown and within a period of time extending, is imaged with for example energy of monitoring process (motion in environment), propagation (growth and division) and morphologic variation (size and shape) under the condition of controlling.Owing to the length of experiment and the view data of the enormous amount that produces, for example fissional duration of extracting parameter and between time may be tediously long task.This is for wherein multiple samples are especially true by the high-throughout application of while imaging.Therefore, there are the needs to can automatically extracting the image analysis software of desired information.

A kind of mode of assessment embryo viablity is the amount of " cytoactive " in measurement image.This can complete by the image and their pixel value of comparison that obtain continuous pairs simply.More particularly, in order to measure the amount of cytoactive of each new images, calculate new images (being labeled as I) and image before (being labeled as I ') between picture element density squared difference with (SSD), whole all overlapping pixel i:

In order to reduce noise, first image can come level and smooth with Gaussian filter.Figure 28 shows the curve map of the single embryo cytoactive of the 1st day to the 3rd day.As shown, have corresponding to 1 cell to 2 cell division in human embryos, 2 cell to 4 cell divisions and the fissional sharp-pointed peak of 4 tenuigenin 8.The width at peak represents the fissional duration.

One of limitation of the method is that SSD measures active amount in measurement image only and for example embryo movement of event (such as displacement or rotation) looking can be closely similar with cell division.To carry out image registration before calculating SSD for a kind of solution of this problem.Image registration be find geometric relationship between two images in case by them in the process of identical coordinate system alignment and can use multiple different technology to complete.For example, can use a kind of modification of Levenberg-Marquardt iteration non-linear process, it is by minimizing the SSD of overlapping picture element density by image registration.LM algorithm uses 3x3 homography matrix to transform pixel site:

Wherein object pixel site x ' and y ' are standardized as:

Therefore

The rational selection that homography matrix can be used in multiple image conversion and this application will be that rigid body (Euclid) transforms.This will shift and Plane Rotation (along photograph arbor) in embryo's image calibration.But vague generalization and use affined transformation are possible a little, it allows image phase poor.This vague generalization is depended on the signal that effort is measured and may is or may not expect.Therefore equation of motion is:

x′＝h ₀x+h ₁y+h ₂

y′＝h ₃x+h ₄y+h ₅

First LM algorithm uses chain rule to calculate e for unknown kinetic parameters h _kpartial derivative:

For affine motion parameter, these partial derivatives are:

Subsequently, use these partial derivatives, LM algorithm is by adding the gradient vector b (in true several destination aggregation (mda)s of big or small 6x1) that calculates approximate Hessian matrix A (in true several destination aggregation (mda)s of big or small 6x6) and weighting from the contribution of each pixel:

Finally, upgrade kinematic parameter by the motion that adds increase:

ΔH＝(A+λI) ^-1b，

Step-length and I that wherein constant λ adjustment movement is upgraded are unit matrixs.

In each iteration of algorithm, according to upgraded locomotion evaluation by the first scalloping and by calculating the SSD of the picture element density in overlapping region and the second image ratio.Embryo movement between this application hypothesis consecutive image is very little and therefore only carries out the iteration of little fixed amount.Figure 28 shows not to be had (28A) and has (28B) every pair of image to be carried out to the curve map of the cytoactive of image registration.Because the formula of variance of Levenberg-Marquardt program is SSD, simply the residual error of calibration is at every turn drawn.Figure 29 has compared the curve map of the cytoactive of normal and undesired embryonic development.At the 3rd day, at embryologist's morphologic point of assessment conventionally, embryo seemed similar and is considered to possibly great-hearted.But their cytoactive curve map is but different greatly, because an embryo in embryo experiences typical a series of cell divisions, other embryos are split into many cells and fragmentation from 1 cell stage.As expected, the embryo who has a normal activity curve map finally reached blastocyst before the 5.5th day.

Before SSD in calculating pixel density, can use other types image registration.This for example comprises that intercorrelation, standardization intercorrelation, mutual phase place are relevant, interactive information, feature detection and tracking, the conversion of yardstick invariant features (SIFT), optical flow method and Gradient Descent.Before registration, for example feature of image preprocessing or contrast enhancing can be or can not be desired.

For assessment of the model of embryo's viablity

The imaging that Figure 13 shows to be correlated with and analysis of molecules are the model that basic human embryos are grown.Shown is the timeline that is developed to blastocyst from zygote, comprises for predicting and is successfully developed to the crucial blink of blastocyst and the chart of embryonic development.As graphic, the bright human embryos of main molecules tables of data start life with heredity from the egg mother cell RNA of mother's different sets.Being integrated into of this RNA is kept by special RNA supervisory routine in ovum and packaging correctly.After fertilization, the degraded of the maternal RNA of the specific sub-set of ovum (ESSP1: embryonic period, embryonic phase specific pattern 1) must be started and degraded to embryo's conversion with egg mother cell.Meanwhile, other RNA continue to be averagely allocated to ideally each blastomere (ESSP4) with growing.The successful degraded of RNA and distribute the transcribing and finish from the embryonic gene group activation (EGA) of master mode and ESSP2 gene with cell.It should be noted that during cleavage division, embryo's blastomere can be stagnated or carry out independently.In embryo, the result of cell autonomous development is that single blastomere can be stagnated or advance and along with 8 cell stages advance to morula stage, and blastocyst quality will be stagnated or the impact of the cell number that exceedes 8 cells that advances.Imaging data confirms to have to be predicted successfully or critical period of failed growth: cytokinesis for the first time, cleavage division for the second time and for the second time and the synchronism of cleavage division for the third time.Cell tracker algorithm and the software before these parameters can be used, described are measured automatically.Described system and method can be used for crucial imaging predictor diagnosis embryo outcome and can allow to shift grow in the less embryo of (before EGA) early.Automatically and the comparison of manual image analysis.

Figure 34 shows 14 embryos' the automated image analysis of set and the comparison of manual image analysis.Embryo 1 to 10 (as institute's mark on curve map) reaches blastocyst stage, has different morphology.Embryo's 11 to 14 stagnations and do not reach blastocyst stage.The comparison of the duration of Figure 34 A display measurement cytokinesis for the first time, and Figure 34 B display measurement is for the first time and the comparison of the time between mitosis for the second time.As directed, two methods demonstrate good consistance conventionally.A small amount of deviation of the duration of cytokinesis for the first time expects, because as previously discussed, the automatic analysis that they are attributable to us is extended and produced approximate value by measurement.In several examples, the duration of cytokinesis and for the first time and for the second time time between mitosis automatically and between manual analyzing, have larger inconsistent.This appears on several abnormal embryos and is caused by the abnormal behaviour that is manually difficult to characterize and is difficult to automatic tracing.For this group embryo with only use two standards (duration of cytokinesis for the first time and for the first time and for the second time time between mitosis), automatic algorithms has zero false positive.In this IVF program that must be avoided in false positive, will be very important.Manual image analysis has a false negative (embryo 9) and automatic algorithms has two false negatives (embryo 9 and 10).But although embryo 9 and 10 technically reaches embryonic period, embryonic phase, they show bad morphology and will be the not good candidates shifting compared with other blastocyst.For manual image analysis, embryo 14 will be taking these two standards as basic false positive and need the 3rd parameter of duration between mitosis for the second time and for the third time to provide real feminine gender.But automatic algorithms is only used two standards just to make correct prediction.These results show that our automatic algorithms can successfully predict the difference between blastocyst and the blastocyst of non-blastocyst and different quality.Therefore, determined the situation with good developmental potentiality for multiple embryos, during IVF program, calculating the grade of their relative mass to select 1 or 2 best embryo is possible for shifting.

Aforesaid is only explanation principle of the present invention.Should be understood that, those skilled in the art can design various schemes, although it is not in description or demonstration clearly herein, has realized principle of the present invention and have been included within the spirit and scope of the present invention.In addition, herein, all embodiment of narration and conditional language are intended to help reader understanding's principle of the present invention not to be construed as being limited to these special embodiment and conditions of narrating with the invention concept that artificially improvement prior art is contributed in principle.In addition, narrate herein principle of the present invention, aspect and embodiment with and all statements expections of specific embodiment contain its 26S Proteasome Structure and Function equivalent.In addition, expect that these equivalents comprise current known equivalent and the equivalent of exploitation in the future, that is, do not consider structure and realize any element that identical function is developed.Thereby, scope of the present invention the unexpected exemplary that shows herein and describe that is limited to.But scope and spirit of the present invention are embodied by appending claims.

Claims

1. a method of determining human embryonic developmental potentiality, it comprises in-vitro measurements cell parameters, wherein said cell parameters comprises alone or in combination:

(a) cytokinesis 1;

(b) time interval between the end of cytokinesis 1 and the beginning of cytokinesis 2; And/or

(c) time interval between the initial sum cytokinesis 3 of cytokinesis 2 initial;

And if

(a ') cytokinesis 1 continues 0 minute to 30 minutes;

The time interval between (b ') end of cytokinesis 1 and the beginning of cytokinesis 2 is 8-15 hour; And/or

The time interval between the initial sum cytokinesis 3 of (c ') cytokinesis 2 initial is 0-5 hour,

Determine that described embryo has good developmental potentiality.

2. the method for claim 1, wherein said cell parameters comprises:

(a) cytokinesis 1;

(b) time interval between the end of cytokinesis 1 and the beginning of cytokinesis 2; With

(c) time interval between the initial sum cytokinesis 3 of cytokinesis 2 initial.

3. method as claimed in claim 1 or 2, wherein said cell parameters is measured by time difference microscopy.

4. method as claimed in claim 1 or 2, wherein said cell parameters further comprises the duration of cell cycle 1.

5. method as claimed in claim 4, the wherein described human embryonic good developmental potentiality of the duration of the cell cycle 1 of 20-27 hour instruction.

6. method as claimed in claim 1, wherein said embryo is fertilized to produce by egg mother cell in vitro.

7. method as claimed in claim 6, wherein said egg mother cell is ripe in vitro.

8. method as claimed in claim 7, the egg mother cell of wherein said maturation in vitro supplements by growth factor.

9. method as claimed in claim 1, is wherein measuring before described parameter embryo without freezing.

10. method as claimed in claim 1, wherein before measuring described parameter, described embryo's process is freezing.